Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2002-03-28
2004-01-13
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S283100, C435S286500, C435S286600, C435S287100
Reexamination Certificate
active
06677132
ABSTRACT:
This application is a 371 of PCT/EP00/04289, filed May 12, 2000, which claims priority to Germany 199 21 999.0, filed May 12, 1999.
DESCRIPTION
The invention relates to a device and a method for determining the concentration of organisms, in particular microorganisms, in a fluid.
The determination of the microbe content of fluids, generally expressed in colony-forming units per unit volume, preferably per milliliter (CFU/ml), normally takes place by inoculating nutrient media with the samples which have been taken, and counting the colonies. This procedure is time consuming and costly. In addition, online measurements of the microbe content, that is to say continuous measurements, are not possible.
In online measurement methods, normally only the biological activity of microorganisms is followed. This takes place, for example, by measuring the oxygen or carbon dioxide content, the pH or the concentration of fluorescent metabolic products. However, these methods do not produce a direct correlation, for example on the basis of the oxygen consumption, between metabolic activity and the number of microorganisms present.
Thus, for example, U.S. Pat. No. 5,224,051 describes an apparatus which measures inter alia the content of dissolved oxygen in cooling lubricants in order to obtain information about microorganisms. The apparatus does not, however, measure the oxygen consumption in a closed volume and, consequently, does not produce a correlation between temperature-dependent oxygen consumption and number of microbes in the solution either.
DE 44 29 809 A1 describes a method for the fully automatic control of fermentation processes for the production of alcoholic beverages in order to remove the heat of fermentation resulting from the alcoholic fermentation from the fermentation tanks. This entails measurement, outside the fermentation tanks, of the density of the liquid fermentation substrate, that is to say a metabolite parameter, but not the number of microorganisms involved in the fermentation.
DE 44 15 444 makes use of continuous measurement of the oxygen transfer rate to determine the physiological conditions in microbiological cultures. The oxygen transfer rate is measured from the fall in the oxygen partial pressure in the gas space using a sterilizable oxygen electrode. Direct calculation of the numbers of microbes from the oxygen consumption and the temperature is not disclosed.
U.S. Pat. No. 4,220,715 describes a method for detecting urinary tract infections in urine samples. This entails determination, on the basis of the oxygen consumption, of whether a preset threshold of microorganisms is exceeded in a urine sample. The method thus permits only qualitative and noncontinuous finding of the presence of certain microorganisms.
DE 196 05 753 A1 describes a method with which it is possible to measure the number of microbes per milliliter. However, the method requires very complicated apparatus because the microorganisms are firstly provided with a nutrient medium in order to achieve an optimal metabolism. In subsequent steps of the method, the intermediate and final products of metabolism are concentrated and fed into a chromatography system with detector and computer units.
None of the described methods enables the concentration of organisms, especially microorganisms, in a fluid to be determined accurately in a continuous, rapid and simple manner.
The technical problem on which the present invention is based is thus to provide a method and a device for carrying out the latter, by which continuous determination of the organism concentration in a fluid can take place in a rapid, simple and cost-effective manner, simultaneously achieving high precision.
The present invention solves the technical problem on which it is based by providing a method for the continuous determination of the concentration of organisms, in particular microorganisms, in a fluid which is present in a line section, where at least one data acquisition unit measures the time-dependent change in at least one metabolite parameter in the line filled with fluid, in particular in the part of the line, the line section, having the data acquisition unit or its sensor and the current organism concentration is found from the measurements by calculation based on a predetermined correlation between the change in the at least one metabolite parameter and the organism concentration. The change in the metabolite parameter measured by the data acquisition unit is preferably transferred to a data processing system and analyzed. A container which contains the fluid to be determined is preferably assigned to the line or line section. The line or line section assigned to the container represents a closed system for the fluid present and measured therein, that is to say a system which does not permit direct exchange of metabolites measured in the measurement zone with the—undefined—detour, where the line or the line section is continuously or periodically supplied from the container assigned thereto with fluid to be measured. The fluid from the container is thus generally not removed, for example using a scoop, from the container and transferred to a closed measurement system. On the contrary, the line is preferably in flow communication with the container, that is to say at least one of the openings of the line opens into the container and can receive fluid from there. The fluid present in the line is in a closed or quasi-closed system resulting from the residence time in the line section. Thus, according to the invention, the line section provides a closed system which makes a true measurement of changes in metabolites possible because of the residence time produced in the line section. A closed system is provided according to the invention inside a container or outside a container, it being possible to measure the change in metabolites directly in the fluid. The invention thus relates to a method for the continuous determination of the concentration of organisms in a in a line section, where a container containing the fluid to be measured is assigned to the line section and where the time-dependent change in at least one metabolite parameter in the closed system resulting from the residence time of the fluid in the line section is measured by at least one data acquisition unit. The invention accordingly provides for an organism-containing fluid which is present in a container and in a line preferably assigned thereto to be investigated in the line for a time-dependent, preferably additionally also temperature-dependent, change in at least one metabolite parameter. This entails the time-and, where appropriate, temperature-dependent change in at least one metabolite parameter, which has previously been determined to be suitable for one or more organism species, being determined by a data acquisition unit and the current organism concentration being calculated from the measurements on the basis of a predetermined correlation between the change in the at least one metabolite parameter and the organism concentration. The invention thus preferably provides for analysis, preferably in a data processing system, of the measurements after measuring the change in at least one metabolite parameter, where determination of the current organism concentration is made possible in the analysis by a previously determined computational association, also referred to as correlation hereinafter, between the change in the at least one measured metabolite parameter and the organism concentration. It is possible according to the invention with the aid of correlations found for specific organisms to find the concentration of the organisms as CFU/ml from the metabolite parameters which have been measured in a time-dependent and, where appropriate, temperature-dependent manner. The invention allows quasi- or semi-continuous and continuous, that is to say online, measurement of organism concentrations, in particular microorganism concentrations, or microbes in fluids in an advantageous manner.
The continuous measurement of microbes allowed accordi
Hofler Thomas
Holzhauer Peter
Walitza Eckehard
Fraunhofer-Gesellschaft zur Förderung der Angewandten-Forschung
Leary Louise N.
Ostrolenk Faber Gerb & Soffen, LLP
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