US3858045A - Detection of biological activity - Google Patents

Detection of biological activity Download PDF

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US3858045A
US3858045A US00353953A US35395373A US3858045A US 3858045 A US3858045 A US 3858045A US 00353953 A US00353953 A US 00353953A US 35395373 A US35395373 A US 35395373A US 3858045 A US3858045 A US 3858045A
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/16Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor using radioactive material
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/801Anerobic cultivation
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/8215Microorganisms
    • Y10S435/822Microorganisms using bacteria or actinomycetales
    • Y10S435/874Pseudomonas
    • Y10S435/875Pseudomonas aeruginosa
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/8215Microorganisms
    • Y10S435/822Microorganisms using bacteria or actinomycetales
    • Y10S435/882Staphylococcus
    • Y10S435/884Staphylococcus epidermidis

Definitions

  • Biological activity in blood such as bacteremia (bacteria in blood)
  • bacteremia bacteria in blood
  • Culturing was done manually and, of course, was a tedious procedure that required considerable periods of time until thephysical presence of bacteria, fungi, or other microorganisms could be detected.
  • a general background reading of about five units is frequently encountered. It has also been generally observed when the gas from a sample inoculated with blood is tested, the background reading generally is increased by to 15 units. In other words, even when the blood sample is from a patient who has no microbial infection, the radioactivity will read, on the average, between about 15 and units.
  • the gas from the blood sample of a patient with bacteremia or the like provides an even higher reading. Generally, a reading of about 30 units or more is used as an indication of microorganisms in the blood sample.
  • references to units, herein, is to units of the Bactec instrument. One hundred units are equal'to 0.025 microcuries of C activity.
  • the background reading of the inoculated sample provides a rather low signal-tonoiseratio and a rather high threshold signal level.
  • This invention is directed to the process of detecting biological activity in the blood wherein:
  • a growth medium including a C containing carbon source which is fermentable to produce carbon dioxide is inoculated with a blood sample;
  • the inoculated sample is exposed to conditions conducive to the'occurrence of normal metabolic processes fora period of time sufficient to cause production of carbon dioxide by the fermenting of said source;
  • the improve- I ment comprises lysing mammalian cells present in said blood sample whereby the background noise for the measurement of gaseous C 0 is reduced.
  • the process employs a nutrient medium that contains water, a suitable C containing carbon source, a nitrogen source, minerals and trace elements.
  • Typical C containing carbon sources may be glucose, sucrose, fructose, galactose, mannose, rhamnose or the like, phenylalanine lysine, arginine, or the like, carboxylic acids such as citric acid or the like, glycerol, or urea.
  • Glucose which is readily available, constitutes a preferred material.
  • the level of radioactivity will vary from about 0.1 to about 10 microcuries per 10 ml of medium.
  • the assimilable nitorgen source may be either organic or inorganic, such as nitrates, nitrites, ammonia, urea, amino acids, or the like, while minerals such as chlorides, sulfates or phosphates of calcium, sodium, potassium, magnesium or the like and trace elements such as manganese, iron, zinc, cobalt or the like, may be employed. Vitamins, cofactors or other enrichment agents such as anti-coagulants may also be added if desired.
  • the medium may also include a buffer for pH adjustment and maintenance.
  • the atmosphere above the culture medium can be air, oxygen, or the like if aerobic tests are being conducted, whereas nitrogen, CO or the like can be employed if anaerobic tests are being conducted.
  • the medium is described in detail in the above publications and particularly in U.S. Pat. No. 3,676,679, JLI 61 l, and JLI 612. While the patent indicates that up to 20 percent or more carbohydrate may be employed in the medium, preferred media are said to contain only up to about 0.001 percent carbohydrate and commercial media do contain such small amounts of carbohydrate added as such. In addition, if the media contains peptone, yeast extract, or the like, an additional 1 percent or so of carbohydrates may be present as a component of this additive. The media normally has a dissolved solids content of about 2.5 percent to about 3.5 percent by weight.
  • Vials containing media for aerobic cultures, designated No. 6A, and vials containing media for anaerobic cultures, designated No. 7A, are commercially available from Johnston Laboratories, Inc.
  • a medium suitable for aerobic fermentation may contain tryptic soy broth, hemin, menadione, sodium polyanethol sulfonate, and C-labeled substrates
  • a medium suitable for anaerobic culture may contain tryptic soy broth, yeast extract, hemin, menadione, L-cysteine, sodium polyanethol sulfonate and C-labeled substrates.
  • the 30 ml of medium in the 50 ml vials has a radioactivity of about 1.5 microcuries.
  • Commercial media have a pH of about 7.3.
  • the sample of blood will be added to the medium in amounts ranging from about 0.5 to about 10 percent by volume and the inoculated medium can be cultured under agitation at temperatures between about C and about 60C and most often between about 33C and about 39C.
  • the length of time for the culture will depend, inter alia, on the amount of inoculum employed and the particular microorganism involved. Tests have demonstrated that microorganisms generally will create a positive response in time periods ranging from 1 to 24 hours.
  • the gaseous atmosphere over the culture medium is tested for radioactivity.
  • readings of about 20 to 25 units or more generally will indicate the presence of biological activity in the blood.
  • the thresh old reading will, of course, depend in part on the background noise due to the general surroundings. Such noise can readily be determined by testing the atmosphere from a medium that has not been inoculated.
  • the reading used to indicate the presence of microorganisms may vary somewhat depending on the level of general background noise. When the threshold level has been reached, the sample is considered positive. Negative samples are further incubated and tested again to insure against the presence of slow growing microorganisms.
  • the mammalian cells of the blood sample that is red cells, white cells, and platelets are lysed before substantial incubation has occurred.
  • the cells may be lysed by contacting them with cold water, by contacting them with chemicals or by subjecting them to physical disruption. Lysis desirably is accomplished prior to inoculation but may also take place during the early stages of incubation; e.g., during the first hour or so of incubation.
  • the blood sample is added to water until the cells disrupt.
  • the rate of lysis depends both upon water temperature and dilution, with lower temperatures and greater dilutinons providing better lysis.
  • deionized sterile water at 0C will lyse cells more rapidly than the same water at room temperature. The lower temperature may provide twice the cell disruption as room temperature.
  • a sample dilution of 1:5 will result in more rapid lysis than a sample dilution of lzl.
  • sufficient water will be employed to provide as sample: water weight ratio of at least about 1:], with the maximum amount of water being a matter of choice.
  • Temperatures employed for the lysis most often will range from about 0C to about 40C.
  • Lysis with water may, of course, be carried out as a separate step before inoculation and incubation. Lysis may also be accomplished, however, by adding the blood sample to a dilute culture medium.
  • a normal culture medium may contain about 2.5 3 percent dissolved solids in purified water and this medium does not lyse cells.
  • a medium containing less than about 1 percent dissolved solids will induce lysis to a significant degree.
  • Media containing from about 0.5 percent to about 1 percent dissolved solids are preferred if lysis is to take place in the media.
  • chemicals may be employed and generally are used at low concentrations (e.g., on the order of 0.1 percent) and for short contact periods.
  • Typical chemicals include nonionic detergents, such as saponin, polyoxyethylene sorbitan, and the like.
  • the chemical can be added to the sample as a'separate step, it may be desirable to incorporate the chemical into the syringe employed'to draw the blood sample from the patient.
  • the blood cells are treated with the chemical at a suitable concentration (such as, for example, 0.1 percent by weight) to effect lysis and when the sample from the syringe is added to the culture medium, the sample is diluted and the concentration of the chemical reduced. Care must be taken to avoid too high concentrations, too powerful chemicals, or too long contact times or all cells, including microbial cells, will be substantially lysed.
  • the selection of agents and treating conditions are within the skill of the art. 1
  • the use of physical means to lyse mammalian cells includes, inter alia, ultrasonic vibration, and the like.
  • lysis will be conducted to disrupt sufficient mammalian cells to reduce the background reading due to the sample by at least about 30 percent and, in a particularly preferred embodiment, lysis will be carried out so as to reduce the background reading due to the blood sample by at least about percent. It is evident that lysis should not be so severe as to destroy microbial cells that may be present in the mixture. Mammalian cells are fragile and generally are more sensitive to lysis than bacteria, fungi and the like. Selective lysis of mammalian cells is within the skill of the routineer.
  • EXAMPLE 1 Blood was drawn from 11 patients for use in this experiment. One 3 ml portion of each sample was injected into 30 ml of culture medium in a 50 ml vial, (No. 6A, Johnston Laboratories, Inc.) while a second 3 ml portion was added to 3 ml of sterile water at about 0C to partially lyse the cells. After a few minutes (3-5 minutes) the second portion (now 6 ml of volume) was also added to 30 ml of the same type of culture medium (No. 6A) in a 50 ml vial.
  • the culture medium contained tryptic soy broth, hemin, menadione, sodium polyanethol sulfonate and C -labeled substrates and was characterized by pH of 7.3 i 0.2.
  • the average peak index for the normal blood sample b. the inoculated sample is exposed to conditions was 23.4 units while the average peak index for the conducive to the occurrence of normal metabolic lysed blood sample was 17.0 units.
  • Other tests in fluids processes for a period of time sufficient to cause that contained no cells indicated a general instrumental 25 production of carbon dioxide by the fermenting of background noise level of 10 on the day that the tests s id sour e; and Of this example were conducted.

Abstract

A sample of blood to be tested for the presence of biological activity is added to a suitable growth medium which includes a C14 containing carbon source fermentable to produce gaseous C14O2. After a suitable incubation period, a portion of the gaseous atmosphere above the medium is analyzed for the presence of radioactivity. In the improvement herein described, mammalian cells in the blood sample are lysed in order to reduce background reading when the radioactivity is measured and to provide a better signal-to-noise ratio.

Description

United States Patent [191 Waters [4 1 Dec. 31, 1974 [54] DETECTION OF BIOLOGICAL ACTIVITY 3,676,679 7/1972 Waters 250/380 [76] Inventor: John R. Waters, 801 Stags Head Rd., Towson, Md. 21204 Primary ExaminerHarold A. Dixon [22] Filed: Apr. 24, 1973 21 Appl. No.: 353,953 [57] ABSTRACT A sample of blood to be tested for the presence of biological activity is added to a suitable growth medium [52] US. Cl 250/303, 195/103.5 R, 2255043325, which includes a CH containing Carbon Source fab [51] Int Cl G01 U00 Clzk U04 mentable to produce gaseous C 0 After a suitable incubation period, a portion of the gaseous atmo- [58] Fleld of Search g sphere above the medium is analyzed for the presence of radioactivity. in the improvement herein described, mammalian cells in the blood sample are lysed in [56] References Cned order to reduce background reading when the radio- UNITED STATES PATENTS activity is measured and to provide a better signal-to- 3,172,235 3/1965 Bjorklund 195/143 noise ratio. 3,492,212 1/1970 Searcy 195/100 3,506,402 4/1970 Simon 250/380 7 Claims, N0 Drawings DETECTION or BIOLOGICAL ACTIVITY DESCRIPTION OF THE INVENTION This invention is directed to an improved means for the detection of biological activity in blood.
Biological activity in blood, such as bacteremia (bacteria in blood), in the past, presented a difficult detection problem. Culturing was done manually and, of course, was a tedious procedure that required considerable periods of time until thephysical presence of bacteria, fungi, or other microorganisms could be detected.
More recently, a radiometric technique for the detection of biological activity in the blood has undergone clinical testing and has been adopted for commercial practice. In that method samples of blood are inoculated into a suitable growth medium that includes a C" containing carbon source, the inoculated medium is incubated for a suitable period, and a portion of the gaseous atmosphere is analyzed for C while in the gaseous state. Such process is described, inter alia, in U.S. Pat. No. 3,676,679 issued July I l, 1972; and in the articles Early Detection of Bacterial Growth, with Carbon"-Labeled Glucose," Radiology, 92, No. 1, pp. 154-5 (January 1969); Automated Radiometric Detection of Bacterial Growth in Blood Cultures," J. Labs. Clin. Med., 75, No. 3, pp. 529-34 (March 1970); and Automated Radiometric Detection of Bacteria in 2,967 Blood Cultures, Applied Microbiology, 22, No. 5, pp. 846-849 (November 1971). A commercial instrument for the practice of a rapid, automated process is available under the tradmark Bactec (Johnston Laboratories, Inc.).
In practicing the process for the gaseous detection of the C 0,, a general background reading of about five units is frequently encountered. It has also been generally observed when the gas from a sample inoculated with blood is tested, the background reading generally is increased by to 15 units. In other words, even when the blood sample is from a patient who has no microbial infection, the radioactivity will read, on the average, between about 15 and units. The gas from the blood sample of a patient with bacteremia or the like, of course, provides an even higher reading. Generally, a reading of about 30 units or more is used as an indication of microorganisms in the blood sample.
Reference to units, herein, is to units of the Bactec instrument. One hundred units are equal'to 0.025 microcuries of C activity.
While the described system has proven effective for the rapid detection of biological activity in the blood and is in commercial use, the background reading of the inoculated sample provides a rather low signal-tonoiseratio and a rather high threshold signal level.
It is an object of this invention to increase the signalto-noise ratio when blood is screened for the presence of biological activity.
It is a further object of this invention to reduce the background reading in the gas measurement of the c"o, when blood is screened for the presence of biological activity.
This invention is directed to the process of detecting biological activity in the blood wherein:
a. a growth medium including a C containing carbon source which is fermentable to produce carbon dioxide is inoculated with a blood sample;
b. the inoculated sample is exposed to conditions conducive to the'occurrence of normal metabolic processes fora period of time sufficient to cause production of carbon dioxide by the fermenting of said source; and
c. at least a portion of the gaseous atmosphere from said fermentation is measured for radioactivity to determine the presence of C 0 The improve- I ment, according to the present invention, comprises lysing mammalian cells present in said blood sample whereby the background noise for the measurement of gaseous C 0 is reduced.
The precise reason for the increased background noise due to components in the blood is not fully understood. Whatever the cause, it has been discovered that the background reading is significantly decreased if the mammalian cells are lysed before being allowed to ferment the substrates in the test medium. In some tests, the background noise due to the blood was almost out in half. By decreasing the background noise due to blood, the signal-to-noise ratio is improved and the threshold level for detection of microbes may be reduced.
Further information regarding the process to which this invention relates and the apparatus for carrying out the process is contained, inter alia, in U.S. Pat. No. 3,676,679; Radiology, 92,,No. I, pp. 154-5 (January 1969), J. Labs. Clin. Med., 75, No. 3, pp. 529-34 (March 1970), Applied Microbiology, 22, No. 5, pp. 846-849 (November 1971), JLI 604, JLI 609A, JLI 610A, JLI 611, and JL1612.
Generally, the process employs a nutrient medium that contains water, a suitable C containing carbon source, a nitrogen source, minerals and trace elements. Typical C containing carbon sources may be glucose, sucrose, fructose, galactose, mannose, rhamnose or the like, phenylalanine lysine, arginine, or the like, carboxylic acids such as citric acid or the like, glycerol, or urea. Glucose, which is readily available,, constitutes a preferred material. Generally, the level of radioactivity will vary from about 0.1 to about 10 microcuries per 10 ml of medium. The assimilable nitorgen source may be either organic or inorganic, such as nitrates, nitrites, ammonia, urea, amino acids, or the like, while minerals such as chlorides, sulfates or phosphates of calcium, sodium, potassium, magnesium or the like and trace elements such as manganese, iron, zinc, cobalt or the like, may be employed. Vitamins, cofactors or other enrichment agents such as anti-coagulants may also be added if desired. Finally, the medium may also include a buffer for pH adjustment and maintenance. The atmosphere above the culture medium can be air, oxygen, or the like if aerobic tests are being conducted, whereas nitrogen, CO or the like can be employed if anaerobic tests are being conducted.
The medium is described in detail in the above publications and particularly in U.S. Pat. No. 3,676,679, JLI 61 l, and JLI 612. While the patent indicates that up to 20 percent or more carbohydrate may be employed in the medium, preferred media are said to contain only up to about 0.001 percent carbohydrate and commercial media do contain such small amounts of carbohydrate added as such. In addition, if the media contains peptone, yeast extract, or the like, an additional 1 percent or so of carbohydrates may be present as a component of this additive. The media normally has a dissolved solids content of about 2.5 percent to about 3.5 percent by weight.
Vials containing media for aerobic cultures, designated No. 6A, and vials containing media for anaerobic cultures, designated No. 7A, are commercially available from Johnston Laboratories, Inc. A medium suitable for aerobic fermentation may contain tryptic soy broth, hemin, menadione, sodium polyanethol sulfonate, and C-labeled substrates, while a medium suitable for anaerobic culture may contain tryptic soy broth, yeast extract, hemin, menadione, L-cysteine, sodium polyanethol sulfonate and C-labeled substrates. The 30 ml of medium in the 50 ml vials has a radioactivity of about 1.5 microcuries. Commercial media have a pH of about 7.3.
Most frequently the sample of blood will be added to the medium in amounts ranging from about 0.5 to about 10 percent by volume and the inoculated medium can be cultured under agitation at temperatures between about C and about 60C and most often between about 33C and about 39C. The length of time for the culture will depend, inter alia, on the amount of inoculum employed and the particular microorganism involved. Tests have demonstrated that microorganisms generally will create a positive response in time periods ranging from 1 to 24 hours.
After a suitable period of time, e.g., 8 hours, the gaseous atmosphere over the culture medium is tested for radioactivity. Employing this invention, readings of about 20 to 25 units or more generally will indicate the presence of biological activity in the blood. The thresh old reading will, of course, depend in part on the background noise due to the general surroundings. Such noise can readily be determined by testing the atmosphere from a medium that has not been inoculated. The reading used to indicate the presence of microorganisms may vary somewhat depending on the level of general background noise. When the threshold level has been reached, the sample is considered positive. Negative samples are further incubated and tested again to insure against the presence of slow growing microorganisms.
In accordance with the improvement of this invention, the mammalian cells of the blood sample, that is red cells, white cells, and platelets are lysed before substantial incubation has occurred. The cells may be lysed by contacting them with cold water, by contacting them with chemicals or by subjecting them to physical disruption. Lysis desirably is accomplished prior to inoculation but may also take place during the early stages of incubation; e.g., during the first hour or so of incubation.
In order to lyse cells with water, the blood sample is added to water until the cells disrupt. The rate of lysis depends both upon water temperature and dilution, with lower temperatures and greater dilutinons providing better lysis. For example, deionized sterile water at 0C will lyse cells more rapidly than the same water at room temperature. The lower temperature may provide twice the cell disruption as room temperature. Similarly, a sample dilution of 1:5 will result in more rapid lysis than a sample dilution of lzl. Generally, sufficient water will be employed to provide as sample: water weight ratio of at least about 1:], with the maximum amount of water being a matter of choice. Temperatures employed for the lysis most often will range from about 0C to about 40C.
Lysis with water may, of course, be carried out as a separate step before inoculation and incubation. Lysis may also be accomplished, however, by adding the blood sample to a dilute culture medium. A normal culture medium may contain about 2.5 3 percent dissolved solids in purified water and this medium does not lyse cells. A medium containing less than about 1 percent dissolved solids, however, will induce lysis to a significant degree. Media containing from about 0.5 percent to about 1 percent dissolved solids are preferred if lysis is to take place in the media.
As an alternate method of lysis, chemicals may be employed and generally are used at low concentrations (e.g., on the order of 0.1 percent) and for short contact periods. Typical chemicals include nonionic detergents, such as saponin, polyoxyethylene sorbitan, and the like. While the chemical can be added to the sample as a'separate step, it may be desirable to incorporate the chemical into the syringe employed'to draw the blood sample from the patient. In the syringe the blood cells are treated with the chemical at a suitable concentration (such as, for example, 0.1 percent by weight) to effect lysis and when the sample from the syringe is added to the culture medium, the sample is diluted and the concentration of the chemical reduced. Care must be taken to avoid too high concentrations, too powerful chemicals, or too long contact times or all cells, including microbial cells, will be substantially lysed. The selection of agents and treating conditions are within the skill of the art. 1
The use of physical means to lyse mammalian cells includes, inter alia, ultrasonic vibration, and the like.
The above methods to lyse cells are known to the art, and, therefore, will not be described further here.
It is not necessary to effect lysis of all mammalian cells, since the lysis of cells provide progressive improvement as the percentage of cells lysed increases. Preferrably, lysis will be conducted to disrupt sufficient mammalian cells to reduce the background reading due to the sample by at least about 30 percent and, in a particularly preferred embodiment, lysis will be carried out so as to reduce the background reading due to the blood sample by at least about percent. It is evident that lysis should not be so severe as to destroy microbial cells that may be present in the mixture. Mammalian cells are fragile and generally are more sensitive to lysis than bacteria, fungi and the like. Selective lysis of mammalian cells is within the skill of the routineer.
The following examples are included in order to illustrate the practice of this invention. These examples are included for illustrative purposes only and in no way are intended to limit the scope of the invention.
EXAMPLE 1 Blood was drawn from 11 patients for use in this experiment. One 3 ml portion of each sample was injected into 30 ml of culture medium in a 50 ml vial, (No. 6A, Johnston Laboratories, Inc.) while a second 3 ml portion was added to 3 ml of sterile water at about 0C to partially lyse the cells. After a few minutes (3-5 minutes) the second portion (now 6 ml of volume) was also added to 30 ml of the same type of culture medium (No. 6A) in a 50 ml vial. The culture medium contained tryptic soy broth, hemin, menadione, sodium polyanethol sulfonate and C -labeled substrates and was characterized by pH of 7.3 i 0.2.
All samples were cultured at 35C and the radioactivity of the gaseous atmosphere over the culture medium was checked initially at 3-hour intervals and later at 24-hour intervals. Two of the 11 samples proved to contain bacteria, while the remaining nine were sterile. For the nine that were sterile, the maximum value for radioactivity (Growth Index) in the gaseous portion over the medium, as recorded on a Bactec instrument, is shown in Table 1 below:
ment.
Since modification of this invention will be apparent to those skilled in the art, it is intended that this invention be limited only by the scope of the appended claims.
I claim:
1. In the process of detecting biological activity in blood wherein:
0.04 microcuries of C glucose was inoculated with 1 ml of sterile blood, and compared to the same medium containing various concentrations of chemical lysing agents. The results are shown in Table 2 below:
Ps. aeruginosa and Staph. epidermidis were inoculated into .lLl 6A vials and into .lLl 6A vial containing 0.1 percent saponin. The detection times and maximum Growth Index were not affected by this treat- TABLE 1 Patient A B C D E F G H 1 Normal blood (N) 22 20 28 23 23 32 18 Lysed blood (L) l8 l5 I4 24 17 14 I9 12 20 (N L) 2 7 6 4 6 9 l3 4 5 As the above table demonstrates, the lysed blood a. a growth medium including a C containing carsamples in each case provide lower background noise 20 bon source which is fermentable to produce carbon than the normal samples. dioxide is inoculated with a blood sample;
The average peak index for the normal blood sample b. the inoculated sample is exposed to conditions was 23.4 units while the average peak index for the conducive to the occurrence of normal metabolic lysed blood sample was 17.0 units. Other tests in fluids processes for a period of time sufficient to cause that contained no cells indicated a general instrumental 25 production of carbon dioxide by the fermenting of background noise level of 10 on the day that the tests s id sour e; and Of this example were conducted. Therefore, the back- C at least a portion of the gaseous atmosphere from ground noise due to normal blood was said fermentation is measured for radioactivity to Whereas the barikground "Olse for the ysed determine the presence of C O the improvement blood Samples was The treatment of comprising lysing mammalian cells present in said this example, therefore, reduced the background noise blood Sample b f h Sample i exposed fo a due to the blood about y a factor of substantial period of time to conditions conducive EXAMPLE 2 to the occurrence of normal metabolic processes,
. whereby the background noise for the measure- A medium of 10 ml of yp y broth comalmng ment of radioactivity of gaseous CO is reduced.
. duces the background noise due to the blood sample by at least about 30 percent.
7. The process of claim 1 wherein the treatment reduces the background noise due to the blood sample by at least about percent.

Claims (7)

1. IN THE PROCESS OF DETECTING BIOLOGICAL ACTIVITY IN BLOOD WHEREIN: A. A GROWTH MEDIUM INCLUDING A C14 CONTAINING CARBON SOURCE WHICH IS FERMENTABLE TO PRODUCE CARBON DIOXIDE IS INOCULATED WITH A BLOOD SAMPLE; B. THE INOCULATED SAMPLE IS EXPOSED TO CONDITIONS CONDUCIVE TO THE OCCURENCE OF NORMAL METABOLIC PROCESS FOR A PERIOD OF TIME SUFFICIENT TO CAUSE PRODUCTION OF CARBON DIOXIDE BY THE FERMENTING OF SAID SOURCE; AND C. AT LEAST A PORTION OF THE GASEOUS ATMOSPHERE FROM SAID FERMENTATION IS MEASURED FOR RADIOACTIVITY TO DETERMINE THE PRESENCE OF C14O2, THE IMPROVEMENT COMPRISING LYSING MAMMALIAN CELLS PRESENT IN SAID BLOOD SAMPLE BEFORE THE SAMPLE IS EXPOSED FOR A SUBSTANTIAL PERIOD OF TIME TO CONDITIONS CONDUCIVE TO THE OCCURENCE OF NORMAL METABOLIC PROCESSES, WHEREBY THE BACKGROUND NOISE FOR THE MEASUREMENT OF RADIOACTIVITY OF GASEOUS C14O2 IS REDUCED.
2. The process of claim 1 wherein the cells are lysed by mixing the blood with water prior to inoculation of the growth medium.
3. The process of claim 1 wherein the cells are lysed by admixing the blood with a dilute growth medium containing less than about 1 percent by weight of dissolved solids.
4. The process of claim 1 wherein the cells are lysed with a chemical.
5. The process of claim 1 wherein the cells are subjected to physical treatment to lyse them.
6. The process of claim 1 wherein the treatment reduces the background noise due to the blood sample by at least about 30 percent.
7. The process of claim 1 wherein the treatment reduces the background noise due to the blood sample by at least about 50 percent.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3941660A (en) * 1975-01-20 1976-03-02 Jeffrey Mirsky Method and apparatus for detecting micro-organisms
US4526865A (en) * 1981-10-01 1985-07-02 Amb Systems Corp. Microorganism identification technique
US4994378A (en) * 1989-09-08 1991-02-19 Becton, Dickinson And Company Method for reducing blood carbon dioxide background in bacterial media by the addition of micelles of saponin and a phospholipid
US5470711A (en) * 1986-03-26 1995-11-28 Synbiotics Corporation Saliva enhancement reagent for feline leukemia virus elisa

Citations (4)

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US3172235A (en) * 1960-11-26 1965-03-09 Bjorklund Knut Bertil Apparatus for live cell propagation
US3492212A (en) * 1966-12-01 1970-01-27 Hoffmann La Roche Ultrasonic treatment of protein materials
US3506402A (en) * 1964-06-12 1970-04-14 Helmut F Simon Method and device for measuring the radioactivity of 14c- and/or 3h-marked compounds
US3676679A (en) * 1970-04-22 1972-07-11 Johnston Lab Inc Apparatus for detecting biological activity

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3172235A (en) * 1960-11-26 1965-03-09 Bjorklund Knut Bertil Apparatus for live cell propagation
US3506402A (en) * 1964-06-12 1970-04-14 Helmut F Simon Method and device for measuring the radioactivity of 14c- and/or 3h-marked compounds
US3492212A (en) * 1966-12-01 1970-01-27 Hoffmann La Roche Ultrasonic treatment of protein materials
US3676679A (en) * 1970-04-22 1972-07-11 Johnston Lab Inc Apparatus for detecting biological activity

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3941660A (en) * 1975-01-20 1976-03-02 Jeffrey Mirsky Method and apparatus for detecting micro-organisms
US4526865A (en) * 1981-10-01 1985-07-02 Amb Systems Corp. Microorganism identification technique
US5470711A (en) * 1986-03-26 1995-11-28 Synbiotics Corporation Saliva enhancement reagent for feline leukemia virus elisa
US4994378A (en) * 1989-09-08 1991-02-19 Becton, Dickinson And Company Method for reducing blood carbon dioxide background in bacterial media by the addition of micelles of saponin and a phospholipid

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