US3875012A

US3875012A - Apparatus and method for the detection of microbial pathogens

Info

Publication number: US3875012A
Application number: US437890A
Authority: US
Inventors: Gordon L Dorn; Joseph M Hill
Original assignee: Wadley Res Inst & Blood Bank
Current assignee: J K AND SUSIE L WADLEY RESEARCH INSTITUTE AND BLOOD BANK; Wadley Res Inst & Blood Bank
Priority date: 1974-01-30
Filing date: 1974-01-30
Publication date: 1975-04-01
Anticipated expiration: 1992-04-01
Also published as: JPS51105891A; DE2455981C2; JPS5812061B2; GB1475015A; FR2264593A1; IT1029614B; CA1036055A; FR2264593B1; DE2455981A1; AU7511974A

Abstract

An apparatus for the isolation and concentration of microbial pathogens from a sample fluid which comprises an elongated enclosed centrifugation receptacle containing an evacuated space in contact with a sterile aqueous solution of a material having a greater density than the sample fluid but able to selectively receive microbial pathogens from the sample fluid, a first injectable closure on one end thereof and a second injectable closure on its other end, and a sterile end chamber in communication with the second injectable closure which contains a sample fluid treating solution together with an injection needle assembly which when actuated will pass through the second injectable closure means and allow communication between the chamber and the evacuated space within the centrifugation receptacle to thereby allow the treating fluid to flow from the chamber into the evacuated space. Thereafter, the sample fluid can be injected into the evacuated chamber of the centrifugation tube through a needle passing through the first injectable closure and allow the sample fluid to admix with the pretreating solution in the evacuated chamber. The article is then subjected to centrifugation to allow the microbial pathogens to selectively pass into the liquid filter medium.

Description

United States Patent [191 Dorn et a1.

[ 1 Apr. 1,1975

[ APPARATUS AND METHOD FOR THE DETECTION OF MICROBIAL PATHOGENS [75] Inventors: Gordon L. Dorn; Joseph M. Hill,

both of Dallas, Tex.

[73] Assignee: J. K. and Susie L. Wadley Research Institute and Blood Bank, Dallas, Tex.

[22] Filed: Jan. 30, 1974 [21] Appl. No.: 437,890

[52] US. Cl....,. l95/l03.5 R, 128/218 M, 128/272, 206/222, 215/247, 215/D1G. 3, 210/D1G. 23, 233/26, 195/127 Pllllltll) E.\um1'nerAlvin E. Tanenholtz Ans-[slum bli'aminerRobert J. Warden Almrney, Agent. or FirmRichards, Harris & Medlock [57] ABSTRACT An apparatus for the isolation and concentration of microbial pathogens from a sample fluid which comprises an elongated enclosed centrifugation receptacle containing an evacuated space in contact with a sterile aqueous solution of a material having a greater density than the sample fluid but able to selectively receive microbial pathogens from the sample fluid, a first injectable closure on one end thereof and a second injectable closure on its other end, and a sterile end chamber in communication with the second injectable closure which contains a sample fluid treating solution together with an injection needle assembly which when actuated will pass through the second injectable closure means and allow communication between the chamber and the evacuated space within the centrifugation receptacle to thereby allow the treating fluid to flow from the chamber into the evacuated space. Thereafter, the sample fluid can be injected into the evacuated chamber of the centrifugation tube through a needle passing through the first injectable closure and allow the sample fluid to admix with the pretreating solution in the evacuated chamber. The article is then subjected to centrifugation to allow the microbial pathogens to selectively pass into the liquid filter medium.

25 Claims, 6 Drawing Figures Pf-IENTEDAPR 1 I97 FIG. 2

FIG.

FI'G. 5

FIG. 5

i ii/ FIG-4 APPARATUS AND METHOD FOR THE DETECTION OF MICROBIAL PATHOGENS This invention relates to the detection of microbial pathogens. In another aspect, this invention relates to a novel apparatus which is used for admixing a sample fluid containing microbial pathogens with a treating fluid and thereafter selectively extracting the microorganisms from the sample fluid. In still another aspect, this invention relates to a novel method and apparatus for diagnosing septicemia.

Scpticemia, which is the presence of pathogenic microorganisms in the blood, is one of the most serious types of infection encountered. in spite of an armament of antibiotics and fungal drugs, the mortality rate from septicemia is approximately 2571. ln addition, when shock accompanies septicemia, the mortality rate increases to over 60%. Patients who are suffering from debilitating diseases, undergoing major surgery, receiving immunosuppressive drugs. or anticancer medications are particularly prone to septicemia.

Early administration of appropriate antibiotic therapy is important in fighting septicemia. Consequently, it is imperative that the physician known as rapidly possible not only whether the patient has septicemia, but also the identity of the infecting microorganisms, and the susceptibility of the microorganisms to antibiotic agents. Thus, the proper diagnosis of septicemia depends upon very rapid and efficient quantititative analysis of the patients blood. It is imperative during the quantitative analysis of the patient's blood or other body lluid that the sample fluid not be contaminated with pathogens from the laboratory environment.

Three analytical systems have been conventionally utilized to determine the presence of microorganisms in a body fluid. These conventional systems include the liquid broth culture technique. the so-called pour plate method, and the filtration method using a solid matrix filter. Each of these systems has its drawbacks, and none of the system provides a rapid detection of microorganisms in the blood sample. Generally, the liquid broth method is not quantitative, the pour plate method and the filtration method using a solid matrix filter are open systems subject to external contamination, e.g., the introduction of pathogens onto the culture by the laboratory atmosphere and personnel.

Recently, an improved method and apparatus have been developed for determining the presence of microbial organisms within a sample fluid. This improved method is very rapid and quantitative. Such method is disclosed in copending U.S. Pat. application Ser. No. 428135. filed Jan. 9, 1974 (Attorneys docket No. B- 2532C which is a continuation-in-part of US. Pat. application Ser. No. 331,693. filed Feb. l2, l973, and entitled Deteetion ofthe Microbial Pathogens. According to this improved method for detection of microbial pathogens, a sample of body fluid such as blood (preferably a lysed blood sample) is deposited upon a liquid filter medium within a confined, sterile Zone. The liquid filter medium has a density greater than the sample fluid and comprises a sterile aqueous solution which will selectively receive microbial pathogens from the sample fluid. Thereafter, the confined sterile zone is subjected to centrifugation to force the sample fluid against the liquid filter medium and cause microbial pathogens to selectively pass therein and thereby separate from the mass of the body fluid sample. Next, the

liquid filter medium containing the microbial pathogens is separated from the remainder of the sample fluid and portions of the liquid filter medium are subjected to culturing conditions.

The improved method described above does provide a very rapid and efficient procedure for separating microbial pathogens from a sample fluid. However, in the preferred method, the sample fluid is admixed with lysing and anticoagulant agents prior to the time that it is injected into the evacuated centrifugation zone containing the liquid filter medium. Some liquid filter mediums are incompatible with some of the pretreating and/or lysing agents. lt has not heretofore been possible to combine the two materials for long periods in the sterilized centrifugation tube. Therefore, it has been necessary to subject the sample fluid to the possibility of external contamination by the additional mixing step which of course means an additional entry into the closed system.

According to one embodiment of the subject invention, an apparatus is provided for use in the improved method described above which will aseptically introduce the lysing agent into the evacuated centrifugation tube containing the liquid filter medium and thereby allow the blood sample or sample fluid to admix therewith just prior to the centrifugation step wherein the microbial pathogens are selectively removed from the remainder of the sample fluid by the liquid filter medium. The improved apparatus of the subject invention generally comprises an elongated centrifugation tube containing an evacuated centrifugation chamber having a first end and a second end; a first injectable closure means sealably closing the first end thereof and a second injectable closure means sealably closing the second end thereof; and an enclosed end chamber containing a treating fluid such as a lysing agent is affixed on the second end of said elongated centrifugation vessel in communication with said second injectable closure means, said end chamber containing an injection means to aseptically transfer said treatment fluid from said end chamber chamber through said second injectablc closure means to said centrifugation chamber. In a specific embodiment said injection means comprises a tubular stylus positioned for reciprocal movement in said end chamber and has a first opening adjacent its leading end and a second opening in its barrel such that when advanced toward said second injectable closure means in said chamber, the stylus will pass through the second injcctable closure means and its first opening will communicate with the evacuated space while its second opening maintains communication with said end chamber; and means to actuate said stylus means.

In a preferred embodiment of the subject invention, the chamber containing the evacuated space also contains an aqueous filter medium having a greater density than the sample fluid and which will selectively receive the microbial pathogens from the sample fluid, and the end chamber contains an aqueous solution of a lysing agent such as purified saponin extract and optionally other materials such as an anticoagulant and/or an oxygen scavenger such as ascorbic acid and/or a thioglycolate. It is preferred that such materials be sufficiently stable so that they will not decompose or be otherwise deleteriously affected under autoclaving conditions.

In accordance with another preferred embodiment of this invention, the above-described centrifugation and mixing apparatus is provided with a syringe means for removing the aqueous filter solution from the evacuated chamber which includes a stylus sufficiently long to only penetrate the thickness of the first injectible closure means and not to materially pass into said aqueous filter medium.

This invention can be more easily understood from a study of the drawings in which:

FIG. 1 is a sectional view of a preferred centrifugation and mixing apparatus of the subject invention;

FIG. 2 is a sectional view of the apparatus of FIG. 1 showing the stylus in the upper chamber in actuated position;

FIG. 3 is a sectional view of the apparatus in FIG. 1 showing a sample fluid being injected therein through the bottom closure:

FIG. 4 is a sectional view of the apparatus of FIG. 3 showing the aqueous filter medium being removed via a needle passing through the lower closure;

FIG. 5 is a sectional view of another embodiment of the subject invention; and

FIG. 6 is a sectional view of the embodiment of FIG.

5 showing the stylus in actuated position.

The improved centrifugation and mixing apparatus of the subject invention provides an improved means for carrying out the method of detecting microbial pathogens which is set forth in copending patent application Ser. No. 428,135 filed Jan. 9, I974, (Attorneys docket No. B2532C) which is a continuation-in-part of US Pat. application Ser. No. 33l ,693, filed Feb. 12, I973, and such disclosure is herein incorporated by reference into the subject application.

Now specifically referring to the drawings, the novel centrifugation and mixing apparatus as illustrated in FIGS. 1-4, will be described in detail. As shown in FIG. 1. the mixing and centrifugation apparatus 10 comprises an elongated, tubular centrifugation vessel 12, having an injectable closure member 14 which sealably closes the lower end thereof, and an injectable closure member 16 which sealably closes the upper end thereof. Furthermore, the upper end of injeetable closure member 16 is enclosed by cap 18. As shown, cap 18 carries screw threads 20 which operatively engage screw threads 22 positioned around the upper periphery of centrifugation vessel 12.

Cap 18 generally comprises a cylindrical body carrying screw threads 20 on the inside periphery of the opening thereof adjacent shoulder 24 which receives the outer rim ofinjectable closure member 16. Furthermore, the outer edge of shoulder 24 forms the opening of cavity 26, which is preferably a short cylindrical shape. The upper end of cavity 26 is enclosed by end wall 28. Rod 30 of push button member 32 extends through aperture 33 in end wall 28. Furthermore, rod 30 carries tubular stylus 34 on its leading end. Tubular stylus 34 comprises a sharp, beveled end 36 and an aperture 38 through its side wall as shown in the drawing. Furthermore, a resilient washer is positioned snugly around the upper end of tubular stylus 34 in a manner to seal the interior of cavity 26 above aperture 38 of tubular stylus 34.

Centrifugal vessel 12 can be made of glass or hard plastic such as polycarbonate or polypropylene and cap 18 can be made of a hard plastic. Injectible closure member 14 can comprise a rubber'self-scaling stopper. The leading end of injectable closure member 14 carries a frustoconical recess 14a. Injectable web 14h forms the bottom of the frusto-conical recess 14a. In;

jectable closure member 16 can also comprise an injectable rubber self-sealing stopper. The top of injectable closure member 16 carries a recess 16a, and the leading end of injectable closure member 16 carries a recess 16b.

Recesses

16a and 16b are separated by injectable web 16c. If desired,

injectable closure members

14 and 16 can be of the same basic configuration. It is only desirable that in jectable closure member 16 carry recess 16a on its top portion and that injcctable closure member 14 carry a frusto conical recess 14a within the leading end thereof. The sterile contents of centrifugation vessel 12 comprises liquid filter medium 42 and an evacuated space 44 which may be a complete or a partial vacuum. Space 44 is maintained at a lower than atmospheric pressure at a predetermined value so that the centrifugation vessel can receive a known amount of liquid by injection through

injectable closure members

14 and 16 without excessive pressure being built up within the interior thereof which could cause

injectable closure members

14 and 16 to become dislodged from the openings within centrifugation vessel [2.

The liquid filter medium 42 can be of any of the liquid filter media set forth in said copending patent application for detecting microbial pathogens and generally, comprises an aqueous solution of any solute which is nontoxic to the microbial organisms being suspended,'

and has a density sufficient high to suspend red and white blood cells or blood cell debris. The solute is preferably nonionic. Thus, 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. In addition, 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 42 include the sugars such as sucrose, glucose, maltose, fructose, manitol, sorbitol, and the like. Generally, liquid filter medium 42 should be at least about 40 wt 7( of the sugar and can contain the sugar up until the saturation limit thereof. Preferably, the sugars are contained within liquid filter medium 42 in the range of from about 50 wt 71 thereof. Generally, the sugars, and especially sucrose, are preferred solutes for liquid filter medium 42 because the liquid filter medium can be maintained at a physiological pH, i.e., 6.0-7.0 and when combined with gelatin, they can be autoclaved. Any solute can be used in the scope of this invention so long as the resulting solution is more dense than red blood cells and red blood cell debris, and is nontoxic to the microbial pathogens. Other suitable such materials include a chemical commonly known as Hypaque sodium, C H I N NaO (3,5 -diacetamido-2,4,6- triidobenzoic acid sodium salt). This material can be utilized in aqueous solution in the same concentration as the sugar as described above. Another class of solutes which can be used to form the aqueous 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 have a pour size small enough to preclude red cells or red cell debris but large enough to pass microbial pathogens.

An example ofa 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 about 500.000, and an intrinsic viscosity of about 0.1 7dl/g, a specific rotation lal 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 FICOLL by Pharmacia Fine Chemicals, Inc. 800 Centennial Avenue, Piscastaway, NJ. Another such polymer which can be used in the scope of this invention is dextran, having a weight average molecular weight in the range of 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 have microporous openings throughout the solubilized network in the range of from about 1 micron to 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 wt "/2 and more preferably from about to about wt 7r thereof.

It is to be understood that the term thermally sensitive gelling agent is meant any agent which will gel the aqueous solution of filter medium 42 at a temperature generally lower than room temperature but yet will liquefy at higher temperatures which are nondeleterious to the microbial pathogens. c.g., lower than about 50C and generally no higher than about 42C. Suitable thermosensitive gelling agents include any such gelling agent which is nondeleterious to the solution or to the sample being analyzed. Examples of suitable such materials include gelatins, i.e., the proteins obtained from collagen by boiling skin, ligaments, tendons, bones, and the like in water. Any suitable amount of thermally sensitive gelling agent can be utilized, e.g., about 0.5 to about 5 wt Z of filter medium 42.

Furthermore. it should be noted that cavity 26 can be any convenient shape and preferably has a round cross sectional area and is a short cylindrical shape and is generally in alignment with recess 16a ofinjectable closure member 16. Thus, tubular stylus 34 which is carried by rod 30 of push button member 32 will axially move within cavity 26, and resilient washer will maintain the upper portion of cavity 26 sealed at a point above aperture 38 of tubular stylus 34. Furthermore. it is noted that the spacing between aperture 38 and beveled end 36 of tubular stylus 34 is slightly greater than the thickness of injectable web 16c such that when push button 32 is fully extended, the opening adjacent beveled and 36 will pass through injectable web I6c and be positioned within space 44 ofcentrifugation vessel 12 while aperture 38 will communicate with cavity 26 as illustrated in FIG. 2. This position of tubular stylus 34 will allow liquid 46 which is contained within or carried by recess 16a to pass into centrifugation vessel 12 by action of the lower pressure within space 44 and pass via aperture 38, the tubular body of stylus 34 and opening adjacent bevel point 16a.

Treating solution 46 can contain any suitable ingredient or ingredients with which it is desired to treat the sample fluid before microbial pathogens are separated therefrom. In accordance with a specific embodiment ofthe subject invention. treating solution 46 comprises an aqueous solution of a lysing agent for blood. Any suitable lysing agent can be utilized in the aqueous solution which is nontoxic to microorganisms. A suitable such lysing agent is a nontoxic aqueous solution of saponin. It must be noted that many saponins are thought to be toxic to microbial pathogens. However, as set forth in copending application Ser. No. 423,447. filed DEC. 10, I973 (Attorneys Docket B-2774) entitled DETOXIFICATION OF SAPONINS, which is herein incorporated by reference into this application, a new method is disclosed for removing the toxic ingredients from the heretofore thought to be toxic saponins. In general, the toxic saponin material can be detoxified in accordance with the invention set forth in this copending patent application and the resulting purified material used in the scope of this invention. Some commercially available saponin preparations are nontoxic, and of course, the materials can also be used in the scope of the subject invention. In addition, the aqueous solution can contain an anticoagulant and/or an oxygen scavenger. A preferred anticoagulant is sodium polyanethol sulfonate (SP8) or Heparin, for exmaple. Sodium polyanethol sulfonate is preferred because it not only acts as an anticoagulant but also inhibits the phagocytic activity of granulocytes and monocytes and the normal antibacterial activity of serum.

Now referring to FIGS. 1-4, the use of the apparatus as illustrated therein will be described in detail. Liquid filter medium 42 can comprise l.5 ml of an aqueous solution containing 50 wt '71 sucrose and 1.5 wt gelatin, for example.

Treating solution 46 can contain any such suitable constituent such as a lysing agent and/or an anticoagulant and/or an oxygen scavenger in any desirable concentration. Any amount of an anticoagulant which is sufficient for the amount of blood sample and any amount of lysing agent sufficient to lyse this blood sample, can be used, for example, 0.3 milliliters of an aqueous solution containing about 12% by weight of nontoxic saponin and about 2% by weight sodium polyanethol sulfonate. Initially, the centrifugation and mixing apparatus 10 is placed in an upright position as illustrated in FIG. 1 to allow liquid filter medium 42 to pass downwardly against injectable closure member 14. Next. apparatus 10 is placed in a suitable cooling unit such as a refrigerator and chilled sufficiently to cause the gelatin to solidify the liquid filter medium 42. For example. the tube can be chilled to 49C. Next, push button member 32 is depressed in a manner illustrated in FIG. 2 to thereby force beveled end 36 of tubular stylus 34 through injectable web such that the opening adjacent beveled end 36 communicates with evacuated space 44 and aperture 38 remains in communication with cavity 26. This action will subject the interior of cavity 26 to the lower pressure maintained within evacuated space 44 and this will therefore result in treating solution 46 passing through aperture 38, the barrel of tubular stylus 34, and from the opening adjacent the beveled end 36 into evacuated space 44 and become deposited as layer 46a upon the solidified liquid filter medium 42.

Next. a sample fluid such as a blood sample (e.g., 8 ml) is obtained within syringe 50 which carries hypodermic needle 52. The blood sample is then injected within the interior of centrifugation vessel 12 in a manner schematically illustrated in FIG. 3. Needle 52 pierces web 14b of injectable closure member 14, passes through the congealed aqueous liquid filter medium 42 and then the plunger of syringe 50. is depressed to deposit blood sample on the congealed liquid filter medium 42 and cause a turbulent mixing with treating solution 46 and form a resulting mixture 48. It is noted that solution 46 can be added to the blood sample after it has been injected into centrifugation vessel 12; however, it is preferred that it be added initially to promote better mixing with the blood sample. The turbulence caused by the blood passing into the evacuated space 44 and turbulently admixing with treating solution 46 will not disturb the liquid filter medium 42 which will remain as a solid bottom layer, as illustrated in FIG. 3. After this is accomplished, cap 18 can be unscrewed and removed from the upper portion of apparatus 10 as illustrated in FIG. 4.

The mixing of the blood sample with the treating solution 46 containing the lysing agent will result in the red blood cells becoming lysed which will therefore minimize the possible trapping effect or erythrocytes and/or lymphocytes. This trapping effect would in general comprise the erythrocytes or lymphocytes becoming stacked on top of the liquid filter medium during the centrifugation step and the stacked cells trapping microbial pathogens as they are passed downwardly during centrifugation and thereby .prevent them for reaching the liquid filter medium. Furthermore, the sodium polyanethol sulfonate within treating solution 46 acts as an anticoagulate and inhibits the phagocytic activity of granulocytes and monoeytes and the normal antibacterial activity of the serum once it becomes admixed with the blood sample.

Next, hypodermic needle 52 is withdrawn from the injcctablc closure member I4 and centrifugation vessel 12 containing the congealed liquid filter medium 42 and the blood sample admixed with treating solution 46 (illustrated as layer 48 in FIG. 3) is heated while in the upright position sufficiently to melt the gelatin and cause the liquid filter medium 42 to liquefy. Centrifugation vessel 12 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. For example, while in the position as illustrated in the drawings, centrifugation vessel 12 can be heated by immersion in a water bath to a temperature set at about 37C-42C. Thus, the liquefication of the gelatin within the liquid filter medium 42 yields a 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 centrifugation vessel I2 into a suitable centrifugation apparatus and subjecting it to sufficient centrifugal force to separate the microbial pathogens from the remaining constituents in the blood sample. The speed and time of centrifugation can vary widely depending upon the construction material of centrifugation vessel 12 and the type of centrifugation apparatus. The centrifugation can be conveniently accomplished by imparting between about 100 and about 6,000 gravities and preferably from about 1400 to 5000 gravities to centrifugation vessel 12. A suitable method would include a swinging bucket centrifuge rotor which imparts between 2000 and 4000 gravities for 10 to minutes to the particular system described in this preferred embodiment.

After centrifugation vessel 12 is subjected to the centrifugation step as described above, a sterile syringe 54 carrying a shortened hypodermic needle 56 is passed through injectable web 14b of injcctable closure member 14 as illustrated in FIG. 4. As illustrated. hypodermic needle 56 carries a stop 56a which will only allow the needle to pierce injectable web 14/; and the beveled end 36 to extend into liquid filter medium 42. After this is accomplished, syringe 54 withdraws liquid filter medium 42 from the interior of centrifugation vessel 12 to leave the residual sample solution mixture 48 therewithin.

It is noted that alternately a longer hypodermic needle can be utilized with syringe 54 and the needle can be passed to the interior of centrifugation vessel 12 to a point slightly past the interface between liquid filter medium 42 and the sample fluid layer 48 and the sample fluid layer initially removed from the tube.

In this instance. the syringe should initially have the plunger retracted and be filled with sterile air in order that the sterile air can be pumped into the interior of centrifugation vessel 12 incrementally as the sample layer is displaced. Thereafter, the liquid filter medium 42 can be removed.

The action of syringe 54 withdrawing liquid filter medium 42 results in the liquid filter medium and the microbial pathogens becoming thoroughly admixed and generally uniformly distributed therein. This liquid filter medium 42 containing the dispersed microbial pathogens is then distributed on suitable bacterial growth media. Suitable such growth media are set forth in said copending patent application entitled DETECTION OF MICROBIAL PATHOGENS.

For example. with l A milliliters of liquid filter medium containing microbial pathogens. one blood agar plate can receive 0.2 milliliters of the medium 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 milliters of the aqueous solution and can be incubated at 37C in an anaerobic environment. Another 0.2 milliliters of the solution can be placed on a sabouraud agar plate and incubated at 25C in an aerobic environment. Another 0.2 milliliters of the solution can be placed on a EMB plate (Eosin methylene blue dye) plate and incubated at 37C in a candle jar. Another 0.5 milliliters of the solution can be placed in a liquid thioglycolate medium and incubated at 37C. The growth media can be checked daily for the presence of colonies. The number of microbial pathogens in l milliliter of 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 final mixture plated. In the general example set forth above. the correction factor is 1.56.

Now referring to FIGS. 5 and 6. a centrifugation and mixing apparatus 10:: is illustrated which shows another embodiment of the subject invention. As shown, centrifugation apparatus 10,, is exactly the same as upparatus 10 except for cap 70. As shown in apparatus 10 cap comprises an enclosed cylindrical cap member having screw threads 72 on the inside walls thereof. and a tubular stylus 74 rigidly attached to the end wall 75 thereof. Tubular stylus 74 comprises the same basic configuration as tubular stylus 34 and comprises a beveled leading end 76 which carries an aperture and a second aperture 78 adjacent its midportion. As shown, with cap 70 in its first position as illustrated in FIG. 5, the beveled end 76 oftubular stylus 74 is positioned within treating solution 46 maintained within recess 16a of injectable closure member 16. in this position. it is preferred that a plastic strip 80 be positioned around the lower outside lip of cap 70. Plastic strip 80 can comprise a thermoplastic or thermosctting material which will deform such as by heat to conform to the lower outside lip of cap 70 and lower outside screw threads 22 carried by centrifugation vessel 12. This will assure that the closure member 70 will be maintained in its first position illustrated in FIG. 5 during storage and handling. lf it is desired to deposit the treating solution 46 upon liquid filter medium 42, cap 70 is then screwed downwardly to thereby deform strip 80 and force the beveled end 76 of tubular stylus 74 through the injectable closure member 16 as illustrated in FIG. 6. This will thereby allow communication between the cavity formed between recess 16a of injcctablc closure member 16 and end wall 75 and the evacuated space 44 and thereby cause treating fluid 46 to pass through aperture 78 through tubular stylus 74 and into the interior of centrifugation vessel 12.

While this invention has been described in relation to its preferred embodiments, it is to be understood that various modifications thereof will now be apparent to one skilled in the art from reading this specification and it is intended to cover such modifications as fall within the scope of the appended claims.

We claim:

I. An apparatus used for the isolation and concentration of microbial pathogens from a sample fluid comprising:

a. an enclosed centrifugation receptacle having a first end and a second end and containing an evacuated space maintained at a lower than atmospheric pressure adjacent a sterile aqueous solution of a material which is non-toxie to said microbial pathogens and has a greater density than the sample fluid but able to selectively receive microbial pathogens from said sample fluid and also containing a minor effective amount of a thermally sensitive gelling agent:

b. first injectable closure means sealably closing said first end of said elongated eentrifugation receptacle:

c. second injectahle closure means sealably closing the second end ofsaid elongated centrifugation receptacle;

d. a scaled end chamber in alignment with said second injectable closure means on said second end of said elongated centrifugation receptacle and containing a sample treating fluid sealed therewithin; and

e. injection needle means positioned within said sealed end chamber for passing said fluid from said end chamber through said second injectable closure means and into said evacuated space.

2. The apparatus of claim 1 wherein said injection needle means comprises a tubular stylus means positioned within said sealed end chamber in alignment with said second injcctable closure means and containing a first opening adjacent its leading end and a second opening through the sidewall thereof at a spaced distance therefrom. and means to advance said stylus means toward said second injectable closure means to a fully advanced position such that the leading end thereof and said first opening passes through said second injectable closure means and into said evacuated space and the second opening thereof remains in said second chamber.

3. The apparatus of claim 2 wherein said end chamber is formed between said second injectable closure means and a hollow cap means detachably affixed to said second end of said ccntrifugation vessel.

4. The apparatus of claim 3 wherein said cap means is threadably engaged with said second end of said centrifugation vessel.

5. The apparatus of claim 4 wherein said tubular stylus depends from said cap means in alignment in said end chamber with said second injectable closure means and is advanced to its said extended position by rotating said threadably engaged cap on said second end of said centrifugation vessel.

6. The apparatus of claim 4 wherein said tubular stylus is positioned on a plunger means in said end chamber in alignment with said second injectable closure means. I

7. The apparatus of claim 4 wherein said minor effective amount of said thermally sensitive gelling agent is from about 1 to about 5 wt /1 of said aqueous solution.

8. The apparatus of claim 7 wherein said thermally sensitive gelling agent is gelatin.

9. The apparatus of claim 8 wherein said aqueous solution is an aqueous solution of a sugar.

10. The apparatus of claim 9 wherein said sugar is sucrose.

11. The apparatus of claim 10 wherein said aqueous solution contains at least 40 wt /1 of said sucrose.

12. The apparatus of claim 8 wherein said aqueous solution is an aqueous solution of a macromolecular solute having microporous openings throughout its solubilized network, said openings being of sufficient size to selectively pass said pathogens from said sample fluid.

13. The apparatus of claim 12 wherein said polymer is epichlorohydrin-sucrose polymer having a molecular weight in a range of from about 300,000 to about 500,000 and a specific rotation [04],, of 56.5".

14. The apparatus of claim 12 wherein said polymer is dextran having a molecular weight of from about 10,000 to about 2.000.000.

IS. A method of detecting microbial pathogens in a body fluid comprising:

a. providing an aqueous filter solution between first and second injectable zones within a centrifugation tube, said aqueous filter solution containing a thermally sensitive gelling agent and being an aqueous solution of material which is non-toxic to said microbial pathogens and has a greater density than said sample fluid but able to selectively receive microbial pathogens from said sample fluid;

b. positioning said centrifugation tube such that said aqueous filter solution rests on said first injeetable zone and thereafter cooling said centrifugation tube to cause said thermally sensitive gelling agent to solidify said aqueous filter solution;

c. depositing a treatment fluid for said body fluid upon said solidified aqueous filter solution by passing an injection needle through said second injectable zone and thereafter injecting said sample treatment fluid into said centrifugation tube and upon said solidified aqueous filter solution:

d. depositing a sample of said body fluid upon said solidified aqueous filter solution and admixing said body fluid with said treatment fluid by passing a hypodermic needle through said first injectable zone and through said solidified aqueous filter solution and thereafter injecting said body fluid through said hypodermic needle into said centrifugation tube;

e. heating said eentrifugation tube sufficiently to liquefy said thermally sensitive gelling agent and thereby liquefy an aqueous filter solution;

f. subjecting said eentrifugation tube to centrifugation in a manner to force said sample of body fluid against said aqueous filter solution and cause said microbial pathogens to selectively pass therein and thereby separate from the residual mass of said body fluid sample and said treating fluid; and

g. separating said aqueous filter solution containing said microbial pathogen from said residual mass of said body fluid.

16. The method of claim 15 wherein said body fluid is blood and said treatment fluid comprises a blood lysing agent.

17. The method of claim 16 wherein said aqueous filter solution comprises an aqueous solution of a non ionic sugar.

18. The method of claim 17 wherein said nonionie sugar is sucrose.

19. The method ofclaim 17 wherein said aqueous filter solution contains at least about 40 wt of said sugar therewithin.

20. The method of claim 16 wherein said aqueous fil- 21. The method of claim 20 wherein said aqueous filter solution comprises an aqueous solution of a copolymer of sucrose and epichlorohydrin which has a molecular weight in the range of from about 300,000 to 500,000 and a specific rotation [01],, of 56.6.

22. The method of claim 21 wherein said aqueous solution contains from about 10m about 40 weight percent of said copolymer.

23. The method of claim 20 wherein said aqueous filter solution comprises an aqueous solution of dextran which has a molecular weight from about l0,000 to about 2,000,000.

24. The method of claim 23 wherein said dextran is present in said aqueous solution in an amount ranging from about 10 to about 40 weight percent thereof.

25. The method of claim 16 further comprises thoroughly mixing said aqueous filter solution containing said microbial pathogens; plating at least portions of said admixed, aqueous filter solution on a growth media for said microbial pathogens.

@22 2 UNITED STATES PATENT o FICE CERTIFICATE OF CORRECTICN Patent No. 3,875,012 Dated P 19.75

Inventor{s) Gordon L. Born and Joseph M. Hill It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 4, line 27, "sufficient" should be -sufficiently"; line 43; "from about 50 wt should be --from about 40 to about 50 wt Column 6, line 17, "exmaple" should be -example-.

'Column 7, line 21, "for" should be from--.

2 0 Column 12, line 11 (Claim 21), "[d] of 56.6" shouldbe 20 "m1 of 56.6'.

Signed and sealed this lst day of July 1975.

(SEAL) Attest:

' C. MARSHALL DANN RUTH C MASON Commissioner of Patents Attesting Officer and Trademarks

Claims

1. An apparatus used for the isolation and concentration of microbial pathogens from a sample fluid comprising: a. an enclosed centrifugation receptacle having a first end and a second end and containing an evacuated space maintained at a lower than atmospheric pressure adjacent a sterile aqueous solution of a material which is non-toxic to said microbial pathogens and has a greater density than the sample fluid but able to selectively receive microbial pathogens from said sample fluid and also containing a minor effective amount of a thermally sensitive gelling agent; b. first injectable closure means sealably closing said first end of said elongated centrifugation receptacle; c. second injectable closure means sealably closing the second end of said elongated centrifugation receptacle; d. a sealed end chamber in alignment with said second injectable closure means on said second end of said elongated centrifugation receptacle and containing a sample treating fluid sealed therewithin; and e. injection needle means positioned within said sealed end chamber for passing said fluid from said end chamber through said second injectable closure means and into said evacuated space.

2. The apparatus of claim 1 wherein said injection needle means comprises a tubular stylus means positioned within said sealed end chamber in alignment with said second injectable closure means and containing a first opening adjacent its leading end and a second opening through the sidewall thereof at a spaced distance therefrom, and means to advance said stylus means toward said second injectable closure means to a fully advanced position such that the leading end thereof and said first opening passes through said second injectable closure means and into said evacuated space and the second opening thereof remains in said second chamber.

3. The apparatus of claim 2 wherein said end chamber is formed between said second injectable closure means and a hollow cap means detachably affixed to said second end of said centrifugation vessel.

6. The apparatus of claim 4 wherein said tubular stylus is positioned on a plunger means in said end chamber in alignment with said second injectable closure means.

7. The apparatus of claim 4 wherein said minor effective amount of said thermally sensitive gelling agent is from about 1 to about 5 wt % of said aqueous solution.

10. The apparatus of claim 9 wherein said sugar is sucrose.

11. The apparatus of claim 10 wherein said aqueous solution contains at least 40 wt % of said sucrose.

13. The apparatus of claim 12 wherein said polymer is epichlorohydrin-sucrose polymer having a molecular weight in a range of from about 300,000 to about 500,000 and a specific rotation ( Alpha )D20 of + 56.5*.

14. The apparatus of claim 12 wherein said polymer is dextran having a molecular weight of from about 10,000 to about 2,000, 000.

15. A METHOD OF DETECTING MICROBIAL PATHOGEN IN A BODY FLUID COMPRISING: A. PROVIDING AN AQUEOUS FILTER SOLUTION BETWEEN FIRST AND SECOND INJECTABLE ZONES WITHIN A CENTRIFUGATION TUBE, SAID AQUEOUS FILTER SOLUTION CONTAINING A THERMALLY SENSITIVE GELLING AGENT AND BEING AN AQUEOUS SOLUTION OF MATERIAL WHICH IS NON-TOXIC TO SAID SAMPLE FLUID BUT ABLE TO SELEC0009 GREATER DENSITY THAN SAID SAMPLE FLUID BUT ABLE TO SELECTIVELY RECEIVE MICROBIAL PATHOGENS FROM SAID SAMPLE FLUID; B. POSTING SAID CONTRIFUGATION TUBE SUCH THAT SAID AQUEOUS FILTER SOLUTION RESTS ON SAID FIRST INJECTABLE ZONE AND THEREAFTER COOLING SAID CENTRIFUGATION TUBE TO CAUSE SAID THERMALLY SENSITIVE GELLING AGENT TO SOLIDIFY SAID AQUEOUS FILTER SOLUTION; C. DEPOSITING A TREATMENT FLUID FOR SAID BODY FLUID UPON SAID SOLIDIFIED AQUEOUS FILTER SOLUTION BY PASSING AN INJECTION NEEDLE THROUGH SAID SECOND INJECTABLE ZONE AND THEREAFTER INJECTING SAID SAMPLE TREATMENT FLUID INTO SAID CENTRIFUGATION TUBE AND UPON SAID SOLIDIFIED AQUEOUS FILTER SOLUTION; D. DEPOSITING A SAMPLE OF SAID BODY FLUID UPON SAID SOLIDIFIED AQUEOUS FILTER SOLUTION AND ADMIXING SAID BODY FLUID WITH SAID TREATMENT FLUID BY PASSING A HYPODERMIC NEEDLE THROUGH SAID FIRST INJECTABLE ZONE AND THROUGH SAID SOLIDIFIED AQUEOUS FILTER SOLUTION AND THEREAFTER INJECTING SAID BODY FLUID THROUGH SAID HYPODERMIC NEEDLE INTO SAID CENTRIFUGATION TUBE; E. HEATING SAID CENTRIFUGATION TUBE SUFFICIENTLY TO LIQUEFY SAID THERMALLY SENSITIVE GELLING AGENT AND THEREBY LIQUEFY AN AQUEOUS FILTER SOLUTION; F. SUBJECTING SAID CENTRIFUGATION TUBE TO CENTRIFUGATION IN A MANNER TO FORCE SAID SAMPLE OF BODY FLUID AGAINST SAID AQUEOUS FILTER SOLUTION AND CAUSE SAID MICROBIAL PATHOGENS TO SELECTIVELY PASS THEREIN AND THEREBY SEPARATE FROM THE RESIDUAL MASS OF SAID BODY FLUID SAMPLE AND SAID TREATING FLUID; AND G. SEPARATING SAID AQUEOUS FILTER SOLUTION CONTAINING SAID MICROBIAL PATHOGEN FROM SAID RESIDUAL MASS OF SAID BODY FLUID.

17. The method of claim 16 wherein said aqueous filter solution comprises an aqueous solution of a nonionic sugar.

18. The method of claim 17 wherein said nonionic sugar is sucrose.

19. The method of claim 17 wherein said aqueous filter solution contains at least about 40 wt % of said sugar therewithin.

20. The method of claim 16 wherein said aqueous filter solution comprises a sterile aqueous solution of a macromolecular solute having microporous openings throughout its solubilized network, said openings being of sufficient size to selectively pass said pathogens from said sample fluid.

21. The method of claim 20 wherein said aqueous filter solution comprises an aqueous solution of a copolymer of sucrose and epichlorohydrin which has a molecular weight in the range of from about 300,000 to 500,000 and a specific rotation ( Alpha )D20 of 56.6*.

22. The method of claim 21 wherein said aqueous solution contains from about 10 to about 40 weight percent of said copolymer.

23. The method of claim 20 wherein said aqueous filter solution comprises an aqueous solution of dextran which has a molecular weight from about 10,000 to about 2,000,000.