CA1114271A - Assay of gram-negative bacteria - Google Patents
Assay of gram-negative bacteriaInfo
- Publication number
- CA1114271A CA1114271A CA325,901A CA325901A CA1114271A CA 1114271 A CA1114271 A CA 1114271A CA 325901 A CA325901 A CA 325901A CA 1114271 A CA1114271 A CA 1114271A
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- Canada
- Prior art keywords
- bacteria
- active agent
- gram
- liquid phase
- fluid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 241000894006 Bacteria Species 0.000 title claims abstract description 33
- 238000003556 assay Methods 0.000 title claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 22
- 239000013060 biological fluid Substances 0.000 claims abstract description 21
- 239000007791 liquid phase Substances 0.000 claims abstract description 14
- 239000006166 lysate Substances 0.000 claims abstract description 11
- 241000239220 Limulus polyphemus Species 0.000 claims abstract description 8
- 230000001413 cellular effect Effects 0.000 claims abstract description 8
- 239000012530 fluid Substances 0.000 claims abstract description 7
- 230000002934 lysing effect Effects 0.000 claims abstract description 6
- 239000004094 surface-active agent Substances 0.000 claims description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- 210000000601 blood cell Anatomy 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 7
- 210000004369 blood Anatomy 0.000 claims description 6
- 239000008280 blood Substances 0.000 claims description 6
- 238000004062 sedimentation Methods 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 238000010790 dilution Methods 0.000 claims description 4
- 239000012895 dilution Substances 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 239000002510 pyrogen Substances 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 244000005700 microbiome Species 0.000 abstract description 13
- 239000012736 aqueous medium Substances 0.000 abstract description 2
- 239000002158 endotoxin Substances 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 9
- 230000003287 optical effect Effects 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 6
- 239000008188 pellet Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 206010003504 Aspiration Diseases 0.000 description 3
- 208000031729 Bacteremia Diseases 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 239000002028 Biomass Substances 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 241001302191 Polyphemus Species 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- HDFXRQJQZBPDLF-UHFFFAOYSA-L disodium hydrogen carbonate Chemical compound [Na+].[Na+].OC([O-])=O.OC([O-])=O HDFXRQJQZBPDLF-UHFFFAOYSA-L 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- -1 iso-octylphenoxy Chemical group 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 1
- 241000606125 Bacteroides Species 0.000 description 1
- 241000606124 Bacteroides fragilis Species 0.000 description 1
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
- 241000588748 Klebsiella Species 0.000 description 1
- 239000012480 LAL reagent Substances 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- GOOHAUXETOMSMM-UHFFFAOYSA-N Propylene oxide Chemical compound CC1CO1 GOOHAUXETOMSMM-UHFFFAOYSA-N 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 230000000118 anti-neoplastic effect Effects 0.000 description 1
- 229940034982 antineoplastic agent Drugs 0.000 description 1
- 238000001660 aspiration Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009640 blood culture Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000005202 decontamination Methods 0.000 description 1
- 230000003588 decontaminative effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000001879 gelation Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 101150085091 lat-2 gene Proteins 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- UYDLBVPAAFVANX-UHFFFAOYSA-N octylphenoxy polyethoxyethanol Chemical compound CC(C)(C)CC(C)(C)C1=CC=C(OCCOCCOCCOCCO)C=C1 UYDLBVPAAFVANX-UHFFFAOYSA-N 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000001698 pyrogenic effect Effects 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000003828 vacuum filtration Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Abstract of the Disclosure A procedure and kit for assay of Gram-negative bacteria in biological fluids by lysing cells in the fluid at pH 6 to 8, removing cellular debris, sedimenting microorganisms from the liquid phase at 5,000 G or more, incubat-ing the microorganisms with Limulus polyphemus amebocyte lysate in aqueous medium, and measuring the amount of resultant turbidity.
Description
Z`71 This invention relates to a method oE assaying Gramrnegative bacteria in biological fluids and to a kit for conducting such an assay.
It has previously been proposed to employ gelation of the lysate of amebocytes of Limulus polyphemus (L~L) as a test for the presence of bacterial endotoxins in biological fluids, but studies have shown that sevexal pyrogenic compounds other than such endotoxins give positive test results, as reported by Elin et al., J. of Infectious Diseases, Vol. 128, 349-352 (1973). Cther studies have shown that the L~L test for endotoxin in blood samples in many cases fails to correlate with blood cultures showing the presence of a variety of Gram-negative bacteria. M~rtinez-G et al., J. of Infectious Diseases Vol. 127, 102-105 (1973). For these reasons, the I~L test has not been effective for assay of biological fluids such as blood even though it has been quantified for determining biomass of Gram-negative bacteria in sea water by Watson et al., Applied and Environm~ntal Microbiol., Vol. 33, 940-946 (1977).
The present invention is based on the discovery that if Gram-negative bacteria in the biological fluid are separated from extraneous sources of pyrogen as well as fram free endotoxin, the amount of lipopoly-saccharide produced by incubation of the Gram-negative bacteria with LimMlus polyphemus amebocyte lysate can be taken as a quantitative measure of the amount of bacteria present in the specimen fluid. me present invention is particularly useful for assaying Gram~negative bacteria in blood but is also applicable to other biological fluids such as cerebro-spinal fluid, urine, ascites, etc.
The present inven~ion accordingly provides a method of assaying Gram-negative bacteria content in biological fluids containing blood cells which comprises lysing blood cells present in the fluid at pH 6 to 8, separating cellular debris from the bacteria-containing liquid phase, separating the bacteria ., , , . . . . .
from the liquid phase, preferably by subjecting the liquid phase to a force of at least 5,000 G to separate microorganisms from said liquid by sedimentation, washing the separated bacteria to remove pyrogens therefrom, incubating said bacteria with Limulus polyphemus amebocyte lysate, and measuring the amount of turbidity formed during said incubation.
The invention also provides a kit for assay of Gram-negative bac-teria in a biological fluid comprising a container of blood-cell-lysing surface-active agent, a container of mulus polyphemus amebocyte lysate, and one or more standard dilutions containing known amounts of Gram-negative bacteria.
The first step of the process, the lysing of the cells present in the biological fluid at pH 6-8, is preferably brought about by mixing the fluid specimen with an aqueous solution of a surface-active agent; a clear, sparkling lysate is produced in a few minutes on standing at room temperature.
The surface-active agents employed are preferably non-ionic, such as condensates of ethylene oxide with hydrophobic bases formed by condensing propylene oxide;
or alkylphenoxy polyethoxylates such as octylphenoxy polyethoxy ethanol, iso-octylphenoxy polyethoxy ethanol, decylphenoxy polyethoxy ethanol, and the like. Particularly preferred is octylphenoxy polyethoxy (9-10) ethanol sold under the trade mark Triton X-100. The concentration of surface-active agent may vary over a wide range, e.g. from 0.1 to 10% by weight of the biological fluid, preferably from 0.5 to 1.5% by weight. The surface-active agent is preferably first dissolved in water (pyrogen-free) to provide a solution containing 0.01 to 1% by weight of surface-active agent, and the biological fluid is mixed with the aqueous solution in appropriate proportions to provide the desired ratio of surface-active agent to biological fluid. The solution of surface-active agent preferably also contains a buffer such as sodium carbonate-sodium bicarbonate at O.OIM to maintain the pH not only of the solution, but also of the mixture of the solution with the biological fluid, ' G, ,ii ' ;,.
-, . . ' . . ..
: : ' . ' ' .
.. , ', " " 1 '; ''` .: :,, . ', . :
'7~
at 6 to 8.
After lyslng of the cells as described above, the relatively coarse cellular debris is separated from the liquid phase, preferably by filtration.
The filter used must be pyrogen-free and have a pore size sufficiently large ~5 to 60 micrometers, preferably 40-60 micrometers) to pass microorganisms with the liquid phase while retaining the largerparticle size solid cellular debris.
The microorganisms are then separated rom the liquid phase, pre-ferably by subjecting the liquid phase to a force of at least 5000 G, prefer-ably 5000 to 12,000 G, for example by centrifugation, to cause sedimentation and separation o the microorganisms from the liquid phase on to a collection surface such as the surface of a glass or plastic centrifuge tube. Following sedimentation, the supernatant liquid phase is removed, preferably by aspir-ation, and the sedimented microorganisms are washed at least once with pyrogen-free water from which they are again separated by sedimentation. The deposit or pellet of microorganisms is then ready for the next stage of the method.
After aspiration o the supernatant, the washed sedimented micro-organisms are preerably further treated by resuspending in a small quantity o pyrogen-free water and ~iltering through a pyrogen-free filter having a pore size of 0.4 to 0.6 micrometers which retains the microorganisms while permitting residual small partlcles o blood cell stroma to pass through as waste. The microorganisms are then suspended in a suitable aqueous medium such as a pyrogen-free 3% aqueous saline ~sodium chloride) solution to form a suspension of the microorganisms. This suspension Cor an aliquot of the suspension~ is then incubated with Limulus polyphemus amebocyte lysate, pre-ferably a~ a constant temperature between 35 and 45C. for about one hour.
A blank or control procedure is carried out in parallel using sterile 3%
;~, aqueous saline in place of the specimen obtained from the biological fluid, to provide a zero standard for the spectro-photometer~ During the incubation period, ~urbidity develops in the dispersion because of the formation of a precipitate or coagulate) and the amount of this turbidity is determined by measuring optical density by conventional procedures, as described for example by Watson et al., Appliod and Environmental MiGrobiol., Vol. 33, 940-946 tl977). A series of standard solutions containing known amounts of Gram-negative bacteria such as scherichia ~E. coli), Klebsiella CK. pneumonia), Bacteroides ~B. fragilis) or the like, or containing ~nown amounts of endotoxins such as E. coli endotoxin, are also incubated separately with L. polyphemus amebocyte lysate. After subtraction o the optical density values for the blank from those o the unknown specimen samples and from those for the standard solutions, the quantity of Gram-negative bacteria in the unknown can readily be determined by comparing the optical density for the unknown sample with a standard curve constructed from the results obtained with the known standards. When the standard used is an endotoxin, a correct-ion factor is employed to convert the results into biomass of Gram-negative bacteria. The procedure of the present invention provides a sensitivity as low as 20 Gram-negative bacteria per milliliter of biological fluid.
The assay of the present invention is of particular importance not only because of its sensitivity but because it can be completed in a short time, less than 4 hours. The invasion of the bloodstream with Gram-negative bacteria is a serious and often life-threatening complication to many clinical disorders as well as precursor to post-operative surgical sepsis. The ser-iousness of bacteremia is further emphasized by the increasing numbers of patients with immunod~ficient conditions brought on by anti-neoplastic treat-ment, since bacteremia is associated with a high patient mortality. In the light of these facts, the rapid and accurate diagnosis of bacteremia made l~ Z~
possible by the assay of the present invention is an important advance in the art.
The ollowing specific example wi;Ll serve to illustrate more fully the nature of the present invention without acting as a limitation upon its scope.
Example A 4 ml aliquot of a venous blood sample collected in a heparinized syringe by venipuncture after skin decontamination is added to a pyrogen free glass flask containing 36 ml of a lyslng solution consisting of pyrogen-free water containing 0.1% by weight of octylphenoxy polyphenoxy ~9-10~ ethanol ~sold under the trade mark - Triton X-100) and O.OlM sodium carbonate-sodium bicarbonate having a pH of 6-8. After gentle mixing of the contents, the flask is allowed to stand at room temperature until complete sparkling lysis is observed, which occurs in about 3-5 minutes. A known sterile blood sample is treated in the ldentical manner to serve as a control. Each lysed sample is then subjected to vacuum filtration through a 60 ml coarse Allihn ilter-ing tube having a sintered glass filter surface ~40-60 micrometer pore dia-meter).
The filtra~e from each specimen is then divided into two 20 ml.
aliquots which are plaeed in 30 ml pyrogen-free centriuge tubes, covered with anodized aliminum caps and subjected to centrifugation in a T-30 fixed angle rotor at 10,000 G for 15 minutes ln an ultracentrifuge at ambient temperature.
At the completion of centrifugation, the supernatant liquid was removed by careful aspiration leaving an almost invisible pellet composed of micro-organisms and small blood cell fragments on the inner surface of the tube.
The pellet was resuspended in 10 ml of sterile distilled pyrogen-free water and after gentle mixing was again subjected to centrifugation and separation of the supernatant under the same conditions. This washing procedure was .~
- ,:.: : . . ::. :.. :, . :., . : .. :: :.
repeated a second time~
After the second washing and removal of the supernatan~ liquid, the pellet was suspended in 1 ml of a saline solution containing 3% by weight sodium chloride in pyrogen-free water.
Each 1 ml aliquot sample was then transferred to a separate test tube. Into other test tubes, there were introduced as standards 1 ml each o various dilutions in 3% aqueous saline of stock E. coli to provide known concentrations ranging from 0 to 100 organisms/ml. A blank was prepared by adding 1 ml of 3% saline to anothcr test tube.
To each tube there was added 0.2 ml of reconstituted Limulus polyphemus amebocyte lysate obtained from a commercial source. After gentle shaking, all the tubes were incubated in a water bath at 40C for 60 minutes.
After incubation, the tubes were again gently shaken and the optical density of the liquid was read lndividually ln a small volume cuvette a~ 360 nm. The optical density of the blank was subtracted rom the optical density of each unknown and of each standard. A standard curve was drawn from the optical densities of the known standards so that by interpola~ion of the optical density for the unknown sample on the standard curve, the number of organisms present in the unknown could be dete-rmined.
Similar results can be achieved by applying the assay procedure to other biological fluids.
A kit suitable for carrylng out the assay of the present invention can be provided in the form of a container, e.g., a vial containing the cell-lysing agent or solution described above, a container of Limulus amebocyte lysate and one or more standard dilutions of Gram-negative bacteria or Gram-negative endotoxin such as E. coll endotoxln. All apparatus including filters and test tubes and all water employed in the assay must be pyrogen-free.
,.
It has previously been proposed to employ gelation of the lysate of amebocytes of Limulus polyphemus (L~L) as a test for the presence of bacterial endotoxins in biological fluids, but studies have shown that sevexal pyrogenic compounds other than such endotoxins give positive test results, as reported by Elin et al., J. of Infectious Diseases, Vol. 128, 349-352 (1973). Cther studies have shown that the L~L test for endotoxin in blood samples in many cases fails to correlate with blood cultures showing the presence of a variety of Gram-negative bacteria. M~rtinez-G et al., J. of Infectious Diseases Vol. 127, 102-105 (1973). For these reasons, the I~L test has not been effective for assay of biological fluids such as blood even though it has been quantified for determining biomass of Gram-negative bacteria in sea water by Watson et al., Applied and Environm~ntal Microbiol., Vol. 33, 940-946 (1977).
The present invention is based on the discovery that if Gram-negative bacteria in the biological fluid are separated from extraneous sources of pyrogen as well as fram free endotoxin, the amount of lipopoly-saccharide produced by incubation of the Gram-negative bacteria with LimMlus polyphemus amebocyte lysate can be taken as a quantitative measure of the amount of bacteria present in the specimen fluid. me present invention is particularly useful for assaying Gram~negative bacteria in blood but is also applicable to other biological fluids such as cerebro-spinal fluid, urine, ascites, etc.
The present inven~ion accordingly provides a method of assaying Gram-negative bacteria content in biological fluids containing blood cells which comprises lysing blood cells present in the fluid at pH 6 to 8, separating cellular debris from the bacteria-containing liquid phase, separating the bacteria ., , , . . . . .
from the liquid phase, preferably by subjecting the liquid phase to a force of at least 5,000 G to separate microorganisms from said liquid by sedimentation, washing the separated bacteria to remove pyrogens therefrom, incubating said bacteria with Limulus polyphemus amebocyte lysate, and measuring the amount of turbidity formed during said incubation.
The invention also provides a kit for assay of Gram-negative bac-teria in a biological fluid comprising a container of blood-cell-lysing surface-active agent, a container of mulus polyphemus amebocyte lysate, and one or more standard dilutions containing known amounts of Gram-negative bacteria.
The first step of the process, the lysing of the cells present in the biological fluid at pH 6-8, is preferably brought about by mixing the fluid specimen with an aqueous solution of a surface-active agent; a clear, sparkling lysate is produced in a few minutes on standing at room temperature.
The surface-active agents employed are preferably non-ionic, such as condensates of ethylene oxide with hydrophobic bases formed by condensing propylene oxide;
or alkylphenoxy polyethoxylates such as octylphenoxy polyethoxy ethanol, iso-octylphenoxy polyethoxy ethanol, decylphenoxy polyethoxy ethanol, and the like. Particularly preferred is octylphenoxy polyethoxy (9-10) ethanol sold under the trade mark Triton X-100. The concentration of surface-active agent may vary over a wide range, e.g. from 0.1 to 10% by weight of the biological fluid, preferably from 0.5 to 1.5% by weight. The surface-active agent is preferably first dissolved in water (pyrogen-free) to provide a solution containing 0.01 to 1% by weight of surface-active agent, and the biological fluid is mixed with the aqueous solution in appropriate proportions to provide the desired ratio of surface-active agent to biological fluid. The solution of surface-active agent preferably also contains a buffer such as sodium carbonate-sodium bicarbonate at O.OIM to maintain the pH not only of the solution, but also of the mixture of the solution with the biological fluid, ' G, ,ii ' ;,.
-, . . ' . . ..
: : ' . ' ' .
.. , ', " " 1 '; ''` .: :,, . ', . :
'7~
at 6 to 8.
After lyslng of the cells as described above, the relatively coarse cellular debris is separated from the liquid phase, preferably by filtration.
The filter used must be pyrogen-free and have a pore size sufficiently large ~5 to 60 micrometers, preferably 40-60 micrometers) to pass microorganisms with the liquid phase while retaining the largerparticle size solid cellular debris.
The microorganisms are then separated rom the liquid phase, pre-ferably by subjecting the liquid phase to a force of at least 5000 G, prefer-ably 5000 to 12,000 G, for example by centrifugation, to cause sedimentation and separation o the microorganisms from the liquid phase on to a collection surface such as the surface of a glass or plastic centrifuge tube. Following sedimentation, the supernatant liquid phase is removed, preferably by aspir-ation, and the sedimented microorganisms are washed at least once with pyrogen-free water from which they are again separated by sedimentation. The deposit or pellet of microorganisms is then ready for the next stage of the method.
After aspiration o the supernatant, the washed sedimented micro-organisms are preerably further treated by resuspending in a small quantity o pyrogen-free water and ~iltering through a pyrogen-free filter having a pore size of 0.4 to 0.6 micrometers which retains the microorganisms while permitting residual small partlcles o blood cell stroma to pass through as waste. The microorganisms are then suspended in a suitable aqueous medium such as a pyrogen-free 3% aqueous saline ~sodium chloride) solution to form a suspension of the microorganisms. This suspension Cor an aliquot of the suspension~ is then incubated with Limulus polyphemus amebocyte lysate, pre-ferably a~ a constant temperature between 35 and 45C. for about one hour.
A blank or control procedure is carried out in parallel using sterile 3%
;~, aqueous saline in place of the specimen obtained from the biological fluid, to provide a zero standard for the spectro-photometer~ During the incubation period, ~urbidity develops in the dispersion because of the formation of a precipitate or coagulate) and the amount of this turbidity is determined by measuring optical density by conventional procedures, as described for example by Watson et al., Appliod and Environmental MiGrobiol., Vol. 33, 940-946 tl977). A series of standard solutions containing known amounts of Gram-negative bacteria such as scherichia ~E. coli), Klebsiella CK. pneumonia), Bacteroides ~B. fragilis) or the like, or containing ~nown amounts of endotoxins such as E. coli endotoxin, are also incubated separately with L. polyphemus amebocyte lysate. After subtraction o the optical density values for the blank from those o the unknown specimen samples and from those for the standard solutions, the quantity of Gram-negative bacteria in the unknown can readily be determined by comparing the optical density for the unknown sample with a standard curve constructed from the results obtained with the known standards. When the standard used is an endotoxin, a correct-ion factor is employed to convert the results into biomass of Gram-negative bacteria. The procedure of the present invention provides a sensitivity as low as 20 Gram-negative bacteria per milliliter of biological fluid.
The assay of the present invention is of particular importance not only because of its sensitivity but because it can be completed in a short time, less than 4 hours. The invasion of the bloodstream with Gram-negative bacteria is a serious and often life-threatening complication to many clinical disorders as well as precursor to post-operative surgical sepsis. The ser-iousness of bacteremia is further emphasized by the increasing numbers of patients with immunod~ficient conditions brought on by anti-neoplastic treat-ment, since bacteremia is associated with a high patient mortality. In the light of these facts, the rapid and accurate diagnosis of bacteremia made l~ Z~
possible by the assay of the present invention is an important advance in the art.
The ollowing specific example wi;Ll serve to illustrate more fully the nature of the present invention without acting as a limitation upon its scope.
Example A 4 ml aliquot of a venous blood sample collected in a heparinized syringe by venipuncture after skin decontamination is added to a pyrogen free glass flask containing 36 ml of a lyslng solution consisting of pyrogen-free water containing 0.1% by weight of octylphenoxy polyphenoxy ~9-10~ ethanol ~sold under the trade mark - Triton X-100) and O.OlM sodium carbonate-sodium bicarbonate having a pH of 6-8. After gentle mixing of the contents, the flask is allowed to stand at room temperature until complete sparkling lysis is observed, which occurs in about 3-5 minutes. A known sterile blood sample is treated in the ldentical manner to serve as a control. Each lysed sample is then subjected to vacuum filtration through a 60 ml coarse Allihn ilter-ing tube having a sintered glass filter surface ~40-60 micrometer pore dia-meter).
The filtra~e from each specimen is then divided into two 20 ml.
aliquots which are plaeed in 30 ml pyrogen-free centriuge tubes, covered with anodized aliminum caps and subjected to centrifugation in a T-30 fixed angle rotor at 10,000 G for 15 minutes ln an ultracentrifuge at ambient temperature.
At the completion of centrifugation, the supernatant liquid was removed by careful aspiration leaving an almost invisible pellet composed of micro-organisms and small blood cell fragments on the inner surface of the tube.
The pellet was resuspended in 10 ml of sterile distilled pyrogen-free water and after gentle mixing was again subjected to centrifugation and separation of the supernatant under the same conditions. This washing procedure was .~
- ,:.: : . . ::. :.. :, . :., . : .. :: :.
repeated a second time~
After the second washing and removal of the supernatan~ liquid, the pellet was suspended in 1 ml of a saline solution containing 3% by weight sodium chloride in pyrogen-free water.
Each 1 ml aliquot sample was then transferred to a separate test tube. Into other test tubes, there were introduced as standards 1 ml each o various dilutions in 3% aqueous saline of stock E. coli to provide known concentrations ranging from 0 to 100 organisms/ml. A blank was prepared by adding 1 ml of 3% saline to anothcr test tube.
To each tube there was added 0.2 ml of reconstituted Limulus polyphemus amebocyte lysate obtained from a commercial source. After gentle shaking, all the tubes were incubated in a water bath at 40C for 60 minutes.
After incubation, the tubes were again gently shaken and the optical density of the liquid was read lndividually ln a small volume cuvette a~ 360 nm. The optical density of the blank was subtracted rom the optical density of each unknown and of each standard. A standard curve was drawn from the optical densities of the known standards so that by interpola~ion of the optical density for the unknown sample on the standard curve, the number of organisms present in the unknown could be dete-rmined.
Similar results can be achieved by applying the assay procedure to other biological fluids.
A kit suitable for carrylng out the assay of the present invention can be provided in the form of a container, e.g., a vial containing the cell-lysing agent or solution described above, a container of Limulus amebocyte lysate and one or more standard dilutions of Gram-negative bacteria or Gram-negative endotoxin such as E. coll endotoxln. All apparatus including filters and test tubes and all water employed in the assay must be pyrogen-free.
,.
Claims (13)
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method of assaying Gram-negative bacteria content in biological fluids containing blood cells which comprises lysing blood cells present in the fluid at pH 6 to 8, separating cellular debris from the bacteria-containing liquid phase, separating the bacteria from the liquid phase and washing them to remove pyrogens therefrom, incubating said bacteria with Limulus polyphemus amebocyte lysate, and measuring the amount of turbidity formed during said incubation.
2. The method claimed in claim 1 in which the bacteria are separated from the liquid phase by subjecting the liquid phase to a force of at least 5000 G to separate bacteria from said liquid by sedimentation.
3. The method claimed in claim 1 in which the step of lysing the blood cells of the biological fluid comprises mixing the fluid with a surface-active agent.
4. The method claimed in claim 1 in which the cellular debris is separated by filtration through a pyrogen-free filter and the sedimentation of bacteria is carried out by centrifugation at 5000 to 12,000 G.
5. The method claimed in claim 1 in which the biological fluid is blood.
6. The method claimed in claim 5 in which the step of lysing the blood cells of the biological fluid comprises mixing the fluid with a surface-active agent.
7. The method claimed in claim 5 in which the cellular debris is separated by filtration through a pyrogen-free filter and the separation of bacteria is carried out by centrifugation at 5000 to 12,000 G.
8. The method claimed in claim 6 in which the cellular debris is separated by filtration through a pyrogen-free filter and the separation of bacteria is carried out by centrifugation at 5000 to 12,000 G.
9. The method claimed in claim 7 in which the surface-active agent is nonionic.
10. The method as claimed in claim 8 in which the surface-active agent is nonionic.
11. The method as claimed in claim 9 in which the surface-active agent is octylphenoxy polyethoxy (9-10) ethanol.
12. The method as claimed in claim 10 in which the surface-active agent is octylphenoxy polyethoxy (9-10) ethanol.
13. A kit for assay of Gram-negative bacteria in a biological fluid comprising a container of blood-cell-lysing surface-active agent, a container of Limulus polyphemus amebocyte lysate, and one or more standard dilutions containing known amounts of Gram-negative bacteria.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US89829178A | 1978-04-20 | 1978-04-20 | |
| US898,291 | 1978-04-20 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1114271A true CA1114271A (en) | 1981-12-15 |
Family
ID=25409227
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA325,901A Expired CA1114271A (en) | 1978-04-20 | 1979-04-19 | Assay of gram-negative bacteria |
Country Status (9)
| Country | Link |
|---|---|
| JP (1) | JPS54149699A (en) |
| BE (1) | BE875716A (en) |
| CA (1) | CA1114271A (en) |
| CH (1) | CH641493A5 (en) |
| DE (1) | DE2915569A1 (en) |
| FR (1) | FR2423784A1 (en) |
| GB (1) | GB2019563B (en) |
| IT (1) | IT1119718B (en) |
| NL (1) | NL7902983A (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4322217A (en) | 1980-06-27 | 1982-03-30 | Mallinckrodt, Inc. | Process for preparing Limulus lysate |
| US5594113A (en) * | 1988-06-23 | 1997-01-14 | Associates Of Cape Cod, Inc. | Endotoxin binding and neutralizing protein and uses thereof |
| CN104777291B (en) | 2007-10-10 | 2017-09-12 | 普凯尔德诊断技术有限公司 | System for identifying bacterium in urine |
| CN104251911B (en) | 2008-02-05 | 2017-05-31 | 普凯尔德诊断技术有限公司 | System for identifying bacterium in biological sample |
| US10288632B2 (en) | 2009-09-21 | 2019-05-14 | Pocared Diagnostics Ltd. | System for conducting the identification of bacteria in biological samples |
| GB2477752A (en) * | 2010-02-11 | 2011-08-17 | Arab Biotechnology Company | Detection of bacteria |
| JP6279609B2 (en) | 2012-12-11 | 2018-02-14 | ポカード・ディアグノスティクス・リミテッドＰｏｃａｒｅｄ Ｄｉａｇｎｏｓｔｉｃｓ， Ｌｔｄ． | Optical cup with curved bottom |
-
1979
- 1979-04-03 GB GB7911610A patent/GB2019563B/en not_active Expired
- 1979-04-12 IT IT21820/79A patent/IT1119718B/en active
- 1979-04-17 NL NL7902983A patent/NL7902983A/en not_active Application Discontinuation
- 1979-04-18 DE DE19792915569 patent/DE2915569A1/en not_active Withdrawn
- 1979-04-19 CA CA325,901A patent/CA1114271A/en not_active Expired
- 1979-04-19 BE BE0/194719A patent/BE875716A/en not_active IP Right Cessation
- 1979-04-20 CH CH377279A patent/CH641493A5/en not_active IP Right Cessation
- 1979-04-20 FR FR7910134A patent/FR2423784A1/en active Granted
- 1979-04-20 JP JP4888479A patent/JPS54149699A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| DE2915569A1 (en) | 1979-11-08 |
| CH641493A5 (en) | 1984-02-29 |
| GB2019563A (en) | 1979-10-31 |
| BE875716A (en) | 1979-08-16 |
| JPS54149699A (en) | 1979-11-24 |
| FR2423784B3 (en) | 1982-02-26 |
| NL7902983A (en) | 1979-10-23 |
| IT7921820A0 (en) | 1979-04-12 |
| GB2019563B (en) | 1982-07-28 |
| IT1119718B (en) | 1986-03-10 |
| FR2423784A1 (en) | 1979-11-16 |
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