|Publication number||US3928139 A|
|Publication date||Dec 23, 1975|
|Filing date||Jan 9, 1974|
|Priority date||Feb 12, 1973|
|Also published as||CA1021238A, CA1021238A1, DE2406362A1, DE2406362C2|
|Publication number||US 3928139 A, US 3928139A, US-A-3928139, US3928139 A, US3928139A|
|Inventors||Gordon L Dorn|
|Original Assignee||Wadley Res Inst & Blood Bank|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (2), Referenced by (45), Classifications (34)|
|External Links: USPTO, USPTO Assignment, Espacenet|
United States Patent 1191 Dorn [ Dec. 23, 1975 DETECTION OF MICROBIAL PATHOGENS  Inventor: Gordon L. Dorn, Dallas, Tex.
 Assignee: J. K. & Susie L. Wadley Research Institute & Blood Bank, Dallas, Tex.
22 Filed: Nov. 9, 1974 21 Appl. No.: 428,135
Related US. Application Data  Continuation-impart of Ser. No. 331,693, Feb. 12,
 US. Cl l95/l03.5 ,R; 195/127; l95/99  Int. Cl. C12K 1/04  Field of Search l95/l03.5 R, 127, 139,
195/100, 126; 2l0/DIG. 23
 References Cited UNITED STATES PATENTS 4/1955 Carski .L 195/126 1/1963 Baptist l95/lO0X  ABSTRACT A method and apparatus are disclosed which provide for the rapid quantitative detection of microbial pathogens in a sample fluid such as blood. Initially 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.
47 Claims, 10 Drawing Figures .5. Patent Dec. 23, 1975 3,928,139
COOLING HEATING CENTRI FUGATION l DETECTIONOF MICROBIAL PATIIOGENS tively extracting microorganisms from a sample fluid.
In still another aspect, 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. In spite of an armament of antibiotics and fungal drugs, the mortality rate from septicemia isapproximately 25%. In addition, when 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.
Early administration of appropriate antibiotic therapy is important in fighting septicemia. Therefore, it is imperative for the physician to know as rapidly as possible not only whether a patient has septicemia, but also the identity of the infecting microorganisms, and the susceptibility of the microorganisms to antibiotic agents. Thus, proper diagnosis of septicemia depends upon very rapid and e'fficient quantitative analysis of the patients blood.
Conventionally, three analytical systems have been used to determine the presence of microorganisms in a body fluid. Perhaps the most common system is the liquid broth culture technique. In this method, gener ally from to milliliters of blood are added aseptically to a vacuum bottle containing nutrient media and an anaerobic or aerobic gas environment. The bottles are checked daily for visible growth. When growth is observed, or on 2 days and 10 days from theinitiation of the test, a l milliliter sample is drawn and streaked on agar plates. If organisms are present, they will form colonies on these plates in approximately 24 hours.
Another commonly used method is the so-called pour plate method. When using 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. In this system, 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.
ln'addition, in emergency situations wherein the patient has overt acute septicemia, blood samples are subjected to microscopic examination. However, microscopie examination is only used to determine relatively large quantities of bacteria in blood and cannot be utilized for the determination of smaller amounts of microorganisms within the blood.
"None of the first three above-described analytical systems provides a rapid detection of microorganisms in a blood sample. It is noted that death from a serious bloodstream; infection can occur within 24 to 48 hours, and indeed, in many cases occurs before the analytical test procedure is completed. More specifically, the liquid broth method is notquantitative.
r Furthermore, the liquid broth method is susceptible to overgrowth by faster growing microorganisms. In essence, 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. It is necessary for the physician to know the exact number of organisms present in the bloodstream. This will aid him in determining the seriousness of the blood infection and the dosage of the proper antibiotic. 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.
Furthermore,.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.
While 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. First, 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. In addition, 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.
It must be noted that all of the conventional tests are relatively expensive to run. While the filtration techniqueis probably the most expensive, and the liquid technique is probably the least expensive, each of these techniques is relatively expensive, not only in terms of materials, but in the cost of laboratory technician time.
Therefore, 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.
Therefore, 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.
According to one embodiment of this invention, a novel method is provided 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.
According to another embodiment of this invention, a method of detection of microbial pathogens within a sample of body fluid such as blood is provided 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.
According to another embodiment of this invention, a novel apparatus used in the detection of microbial pathogens is provided 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. In a preferred embodiment, 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. In addition, 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.
This invention can be more easily understood from a study of the drawings in which:
FIGS. l-l0 schematically illustrate various steps in the preferred analysis procedure of the subject invention utilizing the novel article of the subject invention.
The preferred procedure of the subject invention will be described in detail'in relation to the quantitative determination of bacteria within a blood sample. It is noted that in cases of suspected septicemia it is extremely important that the physician obtain a very rapid analysis of the blood sample. The analysis will rapidly indicate the type of microbial pathogen or pathogens present in the blood, and the amount of such pathogens in the blood.
Now referring to the drawing, an analysis sequence is schematically depicted illustrating a preferred embodiment of the subject invention. A novel Article 20 utilized in the preferred method of the subject invention is shown in FIG. 1. As shown, 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. Basically, 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. 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 include the sugars such as sucrose, glucose, maltose, fructose, mantiol, sorbitol, and the like. Generally, 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. Preferably, the sugars are contained within the liquid filter medium in a range of from about 40 to about 50 wt. thereof. Generally, 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. Other 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.
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 has a pore size .small enough to preclude red cells or red cell debris but large enough to pass microbial pathogens.
An example of 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. Another suitable such polymer which can be used in the scope of this invention is 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. I
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.
It is to be understood that by the term thermally sensitive gelling agent" is meant 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.
As an,example, 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.
Another 12-14 milliliter evacuate test tube containing 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;
two sterile glass syringes (with plungers retracted) and containing 10 milliliters of sterile air and two 1.5 inch Zl-gauge disposable hypodermic needles,
one 1 milliliten disposable syringe and one 1 inch 2l-gauge disposable hypodermic needle;
one 3 milliliter disposable syringe and 1 inch 21- gauge disposable hypodermic needle;
three. blood agar plates;
' one EMB (eosin methylene blue dye) plate one Sabouraud agar plate;
one tube of thioglycolate medium.
It is noted that with the exception of Article 20 or some equivalent article, various apparatus and culture media can be used to carry out the novel process of the subject invention. It is particularly noted that the culture media set forth above are exemplary only and are generally preferred to be utilized for detecting the most commonly known microbial pathogens. 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.
Thus, while various apparatus can be utilized to practice the method of the subject invention, the above list of apparatus and materials can be conveniently utilized in the scope of this invention in a mannerset forth below.
To utilize 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. Next, the article is placed in a suitable cooling unit such as a refrigerator and chilled sufficiently to cause the gelatin to solidify. For example, the tube can be chilled to 4C in the inverted position as illustrated in FIG. 3. i
Next, 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). 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. However, as set forth in my copending patent application (Attorney's Docket No. B2774), Ser. No. 423,447, filed Dec. l0, 1973, entitled Detoxification of Saponins, which is herein incorporated by reference into this application, I have found a new method for removing the toxic ingredients from the heterofore thought to be toxic saponins. In general, 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. Some commercially available 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. Next, 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.
Next, 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. For example, 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. Thus, 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.
After the tube is subjected to the centrifugation step as set forth in FIG. 6 of the drawing, 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. For example, 7.5 milliliters of the sample residue can be withdrawn as schematically illustrated in FIG. 7.
After the removal of the residual sample fluid, it is next desirable to thoroughly admix aqueous filter solution 25 to ensure that any microbial pathogens which may have been received within the solution are uniformly distributed therein. 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. Next, 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. With the apparatus set forth above, 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.
It should again be noted that the exact procedural steps, apparatus and equipment, and types of culture media utilized in the detailed embodiment set forth above, can vary as desired. For example, any known means can be utilized to admix the blood sample with the anticoagulant and/or lysing agent. Furthermore, in step 7 as set forth in the drawing, 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.
The following examples are given to better facilitate the understanding of this invention and are not intended to limit the scope thereof.
I. 9. EXAMPLE 1 Each of several'8 milliliter samples of sterile blood from healthy blood donors was inoculated with 0.4 milliliters of various concentrations of the human the drawing. Next, samples containing the same human pathogen were deposited both in Articles 20 containing the aqueous FICOLL solution and in Articles 20 containing the aqueous sucrose solution (except for P.
. 5 aerugenosa which was only used in FICOLL solution).
52:2?is;3 3 ?:zfgfifi gzfgkff ggfifil aemgl After each of the articles 20 had received the requisite In each instance, 0.3 milliliters of 12% (by weight).j j 3? l. p :j gr aqueous nontoxic saponiri solution was added to eacha f? Ogen p m a of the samples. Next, each sample was transferred asep- 231 e g t z g tically in a manner set forth in conjunction with the at an t Q s"? a owe f fl i 6 description of the drawings to an evacuated container was then .centn L e g z ig m a such as Article 20, containing a predetermined amount centrliuge fitted an H i e Spee o of the aqueous liquid filter medium. More specifically, cenm i E i g g i z a portion of the Articles 20 which were utilized con- 1e i Set g. m d 8 es e tained 1.5 milliliters of an aqueous solution containing Samp e f Su Jecte a re atlve i uga g 9 -25% of an epichlorohydrinsucrose copolymer havgravmes gravmes g3 a graviing a weight average molecular weight in the range of 4080 grammes and 58 gravmes' i i from about 300,000 to about 500,000, and intrinsic each type, Sample a Sublected at viscosity of about 0.17dl/g, a specific rotation of [111 20 these Vanous Speeds for 20 and minutes of 56.5 and which is sold under the trademark of F1- After centrifugation, 8.0 milliliters of the supernatant COLL by Pharmacia Fine Chemicals, Inc. of Piscataabove the aqueous filter layer was removed with a 10 way, New Jersey, and 1.5 wt gelatin (Eastman Purimilliliter syringe. The remaining contents were vigorfied Pigskin). Another portion of the Articles 20 conously admixed in a vortex mixer for about 16- 2 mintained 1.5 milliliters of an aqueous solution containing 25 utes, and 0.2 milliliter samples of this material were wt sucrose and 1.5 wt gelatin, (Eastman Puriplated in rich agar medium for colony counting. Confied Pigskin), having a pH of 6.5. Each of the articles trolled counts of the suspension used in these samples 20 was inverted and chilled to 4C'in an inverted posiwer made in each instance. The mean percent recovery tion before the sample was added and the sample was" (with standard deviations i.e., s.d.) of pathogens in the added in a manner schematically illustrated in FIG. 4 of samples which are set forth in Tables 1-6 below:
TABLE 1 Aqueous-Ficoll Liquid Filter Medium Centrifuge time of 10 minutes Gradient Material: Ficoll p v l Time: 7 i 10 minutes Recovery I 164g I 650g 1465g v, 2520g 4080g 5860g Organism No. of No. of No. of No. of No. of No. of Sam- Sam- Sam- Sam- Sam- Sams.d. ples s.d. ples s.d.. ples s.d. ples s.d. ples s.d. ples S.aureus 69 :22, 13 92 :44, '1'3 1 107 :46, '12 "-88:26, "13 82 :49, 13' :36; 13 E.ca!i 21 :13, 23 37 :19 24 55 :29 24 56 :24, 23. 52 :24, 21 50 :25, 23
aeruginosa 38 :20, 15 33 :13, 13 64 :17, 15 61 :30, 14 67 :22, 15 67 :16, 14 C.albicans' 81 :28, 14 103 9 14 99 2, 14 70 :27; 14 67 :30, 14' 74 :43, 12
TABLE 2 Aqueous-Sucrose Liquid Filter Medium -Centrifuge-.time of 10 minutes- Gradient Material: Sucrose I I Q Q Time: 10 minutes %Recovery RCF 164g 650 1465g Y T 2 5 20 4080g 5860g Organism Nonof No. of No. of No. of No. of No. of Sam- I H Sam- 'Sam- Sam- Sam- Sams.d. ples s.d. l ples s.d. ples s.d. ples s.d. V ples s.d. ples S.aureus 51 :47, 8 128 :33, 6 77 :36, 8. ..131 :44, 8 109.:54, 8 i .124 :41,. 8 E.coli 18 :13, 12 27.:13, 14 41 :17, 15' 50 :17; 15 63 :20, 15 64 :17, 15 P. aeruginosa C.albicans 49 :33, 6 78 :44, 6, 80. 6 106 :41, 5 78 8, 5 97 :62, 6
TABLE 6 Aqueous-Sucrose Liquid Filter Medium Centrifuge Time of 30 Minutes Gradient Material: Sucrose Time: 30 Minutes Recoveryv RFC 164g 650g l465g 2520g 4080g 5860g Organism o. No. of o. No. No, of N0. of
of Samof Sam- Sam- Sams.d. ples Sam- Sams.d. ples s.d. ples s.d. ples s.d. ples s.d. ples s.d.
S.aureus 82 :45, 7 :56, 6 170 :64, 6 :30, 6 :49, 6 169 :45, 05 148 55 E.coli 44 :24, 14 :26, 12 83 1, 12 7, 13 :32, 12 89 :35, 14 65 89 05 P.aeruginosa C.albicans 82 :41, 6 I12 :47, 6 106 :43, 6 106 :70, 6 110 :39, 6
These results are excellent as compared to the methc. Density-centrifugation method of the subject inods for concentrating and recovering microbial pathogens which are presently available in the art.
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:
Eight and three tenths milliliters of patients blood was introduced into a sterile evacuated tube containing 1.7 milliliters of an aqueous solution containing 0.35 weight percent sodium polyanethol sulfonate, and 0.85 weight percent sodium chloride. 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. After this, the aqueous polymer solution in 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 following conditions: i 1 blood agar plate, 37C, aerobic. Three plates (Sabourauds medium), 25C aerobic. 2 blood agar plates, 37C, anaerobic. Three plates (bloodagar medium), 37C, aerobic. 1 blood agar-plate, 37C, candle jar. v Three plates '(blood agar medium), 37C, anaero- 1 Sabourauds agar plate, 25C, aerobic.
I I l The remainder of the sample was placed in a tube of Three plates (blood agar medium), 37C, candle liquid thyoglycolate medium and incubated at 1 37C. The plates and tube were checked daily for All plates were checked daily for signs of microbial signs of microbial growth. The results of the tests growth. are set forth in Table 7 below:
TABLE 7 Blood Bottle Density Centrifuga- Techni ue Pour Plate Method tion Method Organisms Recovered Day Pure solate Day Pure Isolate Number of Day Pure Number of Obtained Obtained Organisms/ml Isolate Organisms/ml of of Blood Obtained Blood Escherichia coli 3rd 3rd 0.5 negative Aerogenes 4th negative negative B hemolytic Streptococcus 2nd lst 50 lst 450 Stre tomyces ne ative negative 2nd 7 Kle siella and Proteus 5rd not tested lst Proteus l0 Klebsiella I44 Staphylococcus aureus negative not tested 2nd I TABLE 7-continued Blood Bottle Density Centrifuga- Technique Pour Plate Method tion Method Organisms Recovered Day Pure Isolate Day Pure Isolate Number of Day Pure Number of Obtained Obtained Organisms/ml Isolate Organisms/ml of of Blood Obtained Blood Staphylococcus aureus negative not tested 2nd 1 Staphylococcus aureus negative not tested 2nd I Cryptococcus 4th not tested 3rd 1 Cryptococcus 4th not tested 3rd 3 a hemolytic Streptococcus negative not tested 5th Pseudomonas aerugenosa negative not tested 2nd 75 a hemolytic Streptococcus negative negative 4th growth in thio only Escherichia coli and 2nd negative lst E coli 10.5 Pseudomonas aeruginosa P. aerugenosa 3 a hemolytic Streptococcus and negative negative lst 11843 hemolytic Strep. B hemolylic Streptococcus 0.4 Pseudomonas negative negative 1 st 94 Candida 6th 2nd 0.6 2nd 0.5 Staphylococcus aureus 2nd negative negative Enterobacter cloacae 3rd negative negative Pseudomonas 3rd lst 10 1 st l8 Klebsiella Pneumoniae 2nd 49 72 Enlerobacter cloacae 3rd negative negative Klebsiella 2nd lst 0.2 0.4 Escherichia coli 2nd lst 1.2 2nd l.5 Cryptococcus negative negative 3rd 1 .5 l3 hemolytic Streptococcus negative negative 2nd 0.4 a hemolytic Streptococcus negative negative lst Streptococcus 70 Staphylococcus epidermidis negative 2nd 2.5 2nd 2.0 (manitol positive) Klebsiella 2nd negative lst l .5 Pseudomonas 2nd 3rd 0.6 2nd 0.9 Staphylococcus aureus negative lst 0.6 negative Escherichia coli 2nd 2nd 0.5 lst Diplococcus pneumoniae 2% 3% .8 3% .3 Pseudomonas aerugenosa & 6th 3% Pseudomonas 5.1 Escherichia coli Pseudomonas only Pseudomonas only 4 Enterobacter .6 Pseudomonas aerugenosa & 3rd .6 .6 Pseudomonas only Klebsiella 7.5 Escherichia coli negative negative 2nd .4 Escherichia coli 1% 8 7.5 Escherichia coli 1% 26 l9 Escherichia coli 1% l5 l4 Propionibacterium 7 negative negative Candida albicans negative 2nd 0.4 negative Enterobacter cloacae 2nd lst 72 lst 281 Escherichia coli 2nd 2nd 2 lst l7 Staphylococcus aureus negative negative 3rd 1.2 Pseudomonas aerugenosa 3rd lst 1.8 2nd 1.5 Pseudomonas aerugenosa 3rd 2nd 200 2nd 200 Pseudomonas aerugenosa negative 2nd .6 2nd .3 Escherichia coli negative lst .4 negative Pseudomonas aerugenosa 3rd 2nd 22 2nd 20 Escherichia coli 2nd 2nd 4 1st 4.5 Pseudomonas aerugenosa lOth 5th .2 10th .3 Pseudomonas aerugenosa 2nd lst 5 lst 6 Escherichia coli 2nd lst 2.5 lst 3 Enterobacter cloacae 2nd lst 3.2 lst 3.9 Pseudomonas aerugenosa 2nd lst 1 l4 lst 89 Pseudomonas aerugenosa 3rd 2nd .6 2nd .9 Escherichia coli 2nd lst 200 lst 200 In the blood hottle technique the Klebsiella was overgrown by Proteus. *These represent separate isolates from one patient. 'The blood bottle never showed signs of microbial growth. Cryptococcus was recovered from the bottle only after subculture onto Sabouraud medium.
A summary of the data contained in Table 7 is presented in Table 8:
TABLE 8 As illustrated in Table 8, 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. In addition, 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. In addition, as set forth above, 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. 1 V 1 Thus, 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. For example, the above-described 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.
It is noted that 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. In general, 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. These 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. For example, if desired, one can autoclave the sucrosegelatin mixture while manufacturing Article without any undesirable breakdown of the gelatin and precipitation thereof. Furthermore, such 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
' 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 upon reading this specification and it is intended to cover such modifications which fall withinthe scope of the appended claims.
I claim: 1
1. In a methodof detecting the presence of microbial pathogens wherein 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:
. a. depositing said sample of body fluid on a liquid filter. medium in a confined sterile zone, said liquid filter 'medium being nontoxic to said microbial pathogens and having a density greater than said sample of body fluid and thereby able to support said body fluid and selectively receive said pathogens from said body fluid; I
subjecting said confined zone to centrifugation thereby forcing said sample of body fluid against said liquid filter medium and causing substantially all of said microbial pathogens to selectively pass therein and thereby separate from the residual v mass of said sample of body fluid; and
c. separating said liquid filter medium containing substantially all of said microbial pathogens from contact with said residual mass of said sample fluid.
2. The method of claim 1 wherein said liquid filter medium comprises an aqueous solution of a nonionic sugar. 1 v
3. The method of claim 2 wherein said nonionic sugar is sucrose.
4. The method of claim 2 wherein said liquid filter medium contains at least about 40 wt'% of said sugar therewithin.
' 5. The method of claim 4 wherein said body fluid is selected from blood, bone marrow, spinal fluid, pleural fluid and urine.
6. The method of claim 5 wherein said body fluid is blood which has been lysed.
'7. The method of claim 1 wherein said 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.
8. The method of claim 7 wherein said 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".
9. The method of claim-8 wherein said aqueous solution contains from about 10 to about 40 weight percent of'said copolymer.
10. The method ofclaim 9 wherein said body fluid is selected from blood, bone marrow, spinal fluid, pleural fluid and urine. 1
' 11. The method of claim 10 wherein said body fluid is blood which has been lysed.
12. The method .of claim 7 wherein said microporous body comprises an aqueous solution of dextran which has a molecular weight from about 10,000 to about 2,000,000.
13. The method of claim 12 wherein said dextran is present in said aqueous solution in an amount ranging from about 10 to, about 40 weight percent thereof.
14. The method of claim 13 wherein said body fluid is selected from blood, bone marrow, spinal fluid, pleu- 19 ral fluid and urine.
15. The method of claim 14 wherein said body fluid is blood which has been lysed.
16. 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:
a. depositing said sample fluid on a liquid filter medium in a confined sterile zone, said liquid filter medium being nontoxic to said microbial pathogens and having a greater density than said sample fluid and thereby able to support said sample fluid and selectively receive said microbial pathogens therefrom;
b. subjecting said confined zone to centrifugation and thereby forcing said sample fluid against said liquid filter medium, said centrifugation continuing for a sufficient time to cause substantially all of said microbial pathogens to selectively pass into said liquid filter medium and to thereby separate from the residual mass of said sample fluid and said antipathogenic factors;
c. separating said liquid filter medium containing substantially all of said microbial pathogens from contact with said residual mass of said sample fluid containing said antipathogenic factors; and
d. analyzing said liquid filter medium for the presence of said microbial pathogens.
17. The method of claim 16 wherein said liquid filter medium comprises an aqueous solution of sugar.
18. The method of claim 17 wherein said aqueous solution contains at least 40 wt of said sugar.
19. The method of claim 18 wherein said sugar is sucrose.
20. The method of claim 16 wherein said sample fluid is a body fluid.
21. The method of claim 20 wherein said body fluid is selected from blood, bone marrow, spinal fluid, pleural fluid, and urine. t
22. The method of claim 21 wherein said body fluid is blood.
23. The method of claim 16 wherein said 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.
24. The method of claim 23 wherein said analyzing comprises microscopically analyzing said liquid filter medium for microbial pathogens.
25. The method of claim 16 wherein said liquid filter body comprises an aqueous solution of'a polymer selected from the group consisting of sucrose-epichlorohydrin copolymer and dextran.
26. The method of claim 25 wherein said sucrose-epichlorohydrin polymer has a molecular weight in the range of from about 300,000 to about 500,000 and a specific rotation ,, of 56.5.
27. The method of claim 25 wherein said dextran has a molecular weight in the range of from about 10,000
a. providing a sterile liquid filter medium within a sterile evacuated centrifugation tube, said liquid filter medium being nontoxic to said microbial pathogens and having a density greater than said body fluid and thereby able to support said body fluid, but selectively receive said microbial pathogens, said liquid filter medium also containing a minor effective amount of a thermally sensitive gelling agent which upon cooling will solidify said liquid filter member;
b. cooling said centrifugation tube to cause said thermally sensitive gelling agent to solidify said liquid filter medium;
c. depositing a sample of said body fluid upon said solidified liquid filter medium within the evacuated centrifugation tube;
(1. heating said centrifugation tube sufficiently to liquefy said thermally sensitive gelling agent and thereby liquefy said liquid filter medium;
e. subjecting said centrifugation tube to centrifugation in a manner to force said sample of body fluid against said liquid filter medium and cause substantially all of said microbial pathogens to selectively pass therein and thereby separate from the residual mass of said body fluid containing said antipathogenic factors;
f. separating said liquid filter medium containing substantially all of said microbial pathogens from the interior of said centrifugation tube and from said residual mass of said body fluid containing said antipathogenic factors; and
g. analyzing said liquid filter medium for the presence of said microbial pathogens.
30. The method of claim 29 wherein said analyzing comprises thoroughly mixing said liquid filter medium containing said microbial pathogens and plating at least portions of said admixed liquid filter medium on growth media for said microbial pathogens.
31. The method of claim 29 wherein said analyzing comprises microscopically analyzing said liquid filter medium for said microbial pathogens.
32. The method of claim 29 wherein said 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.
33. The method of claim 32 wherein said polymer is said copolymer of sucrose and epichlorohydrin and has a molecular weight in the range of from about 300,000 to about 500,000 and a specific rotation [a] of 56.5".
34. The method of claim 32 wherein said polymer is dextran and has a molecular weight in the range of from about 10,000 to about 2,000,000.
35. The method of claim 32 wherein said thermally sensitive gelling agent is gelatin.
36. The method of claim 29 wherein said 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. i
37. The method of claim 36 wherein said sugar is sucrose.
38. The method of claim 36 wherein said thermally sensitive gelling agent is gelatin.
39. 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.
40. The article of claim 39 wherein said minor effective amount of said thermally sensitive gelling agent is from about 1 to about 5 wt of said aqueous solution.
41. The article of claim 40 whereinsaid thermally sensitive gelling agent is gelatin.
42. The article of claim 41 wherein said aqueous solution is an aqueous solution of a sugar.
43. The article of claim 42 wherein said sugar is sucrose.
44. The article of claim 43 wherein said aqueous solution contains at least 40 wt of said sucrose.
45. The article of claim 41 wherein said 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.
46. The article of claim 45 wherein 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 ,, of 565.
47. The article of claim 45 wherein said macromolecular solute is dextran having a molecular weight of from about 10,000 to about 2,000,000.
Page 1 of 2 222g?" UNITED STATES PATENT OFFICE QER'TIFTCATE QF fiORRECTiGN Q Patent Km 3.928.139 Dated December 23. 1975 1mrentor(s) Gordon L. Dorn It is certified that error appears in the above-identified patent Q and that said Letters Patent are hereby corrected as shown below:
F- Col. 1, lines 64 and 65 [Pg. 3', 'line 19] "microscopie" should be microscopic. p
Col. 2, line 29 [Pg. 4, line 19] "relies upon upon an" should be Q relies upon an'.
Col. 4, line 68 [Pg. 10, .line 111 "mantiol" should be -manitol Col. 5, line 12 (second occurrence) [Pg. 10, line 21] "cells" should be -cell'; 1 0 a line 64 [Pg. 12, line 11] "bonds" should be -bones Col. 6, line 7, [Pg. 12 line 21] "evacuate" should be evacuated.
Col. 7, line 22 [Pg. 15 line 14] "syringe" should be -syringes line 41 (first occurrence) [Pg. 16, line 1] "is" should be to-; line 68 [Pg-. l6,'line 2.4] "200" should be --2000--.
Col. 9, line 20 [Pg. 19,- line 15 "of 56. should be --of +56. 5-
Col. 10, line 27 [Pg. 20, line 19] "wer" should be f -were- Q Table 2 [Page 22] under "650g" last line of column "No. of
Samples","'6," should be -6.
Table 3 [Page 23], under650g" between columns and "No. of Samples" entire column missing, insert s. d.
i 44, 8 i i 18, i 47, Table 4, [Page 24] under "4080g" last line of column "9" should be --l09 Q Table 5 [Page 25], under "4080g", line 2 of column "7" should be -107--.
Q Page 2 of 2- 22 233 UNITED STATES PATENT OFFICE QERTIFECATE 0F CORRECTICN Q Fatent No. 3,928,139 Dated December 23, 1975 lzrventork) Gordon L. Dorn It is certified that error appears in the above-identified patent a and that said Letters Patent are hereby corrected as shown below:
Table 6 [Page 26], under "650g", line 1 of column "05" should be -lO5-;
under "2S20g", column heading "No. Samples" Q should be No. oi samples under "4080g", cdlumn should be s.d. s.d'.
Col. 20, line 10 (Claim 29), "'member" should be medium-.
Bigned and Scaled this twenty-fifth Day of May 1976 [SEAL] Arrest:
RUTH C. MASON C. MARSHALL DA Arresting Officer Commissioner oj'lalenrs and Trqdemarkw
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