RAPID BACTERIA TEST

Abstract

The invention relates to methods and devices for quantitative determination of bacteria.

Claims

1. A method for quantitatively determining bacteria in a sample, comprising the steps of: (a) incubating the sample to be tested together with a labelling reagent that can bind to the bacteria to be determined, wherein the labelling reagent is a labelled bacteriophage, preferably a fluorescence-labelled bacteriophage, a luminescence-labelled bacteriophage or a Raman-labelled bacteriophage, and is used in excess with respect to the bacteria to be detected, (b) removing labelling reagent that has not bound to the bacteria from the bacteria by means of a membrane that is impermeable to the bacteria to be determined and is permeable to the labelling reagent, wherein the bacteria to be determined are collected on the membrane, (c) detecting the labelling reagent bound to the bacteria collected, preferably by means of fluorescence measurement, luminescence measurement and/or Raman measurement, and (d) quantitatively evaluating the measurement signal from step (c).

2. The method according to claim 1, wherein the bacteria are selected from the group consisting of Legionella, salmonella, listeria, coliform germs such as E. coli, and hospital germs such as MRSA.

3. The method according to claim 1, wherein a measuring cell is used for the determination that comprises (i) a first chamber, which optionally contains a store for the labelling reagent, (ii) a second chamber, and (iii) a membrane, which is arranged between the first chamber and the second chamber, the membrane being impermeable to the bacteria to be determined and being permeable to labelling reagent that is not bound to the bacteria to be determined, and once the incubation step has finished, labelling reagent that is not bound to the bacteria to be determined being removed from the first chamber and passed through the membrane into the second chamber.

4. The method according to claim 1, wherein said membrane has a pore diameter of approximately 0.5 to 3 m.

5. The method according to claim 1, wherein the labelling reagent and the sample to be tested are mixed before and/or during the incubation procedure in step (a).

6. The method according to claim 1, wherein the labelling reagent bound to the bacteria is detected in step (c) using a laser with a confocal optic as the light source.

7. The method according to claim 6, wherein the laser is used in an integrated device together with a spectrometer so as to form a detection unit.

8. The method according to claim 1, wherein the quantitative evaluation in step (d) comprises sorting a specific number of bacteria into one or more categories on the basis of predetermined threshold values.

9. The method according to claim 1, wherein at least one reference signal, in particular a reference signal generated by a laser, is used for the quantitative evaluation in step (d).

10. A measuring cell for quantitatively determining bacteria, comprising: (i) a first chamber, which contains a store for a labelling reagent, wherein the labelling reagent is a labelled bacteriophage, preferably a fluorescence-labelled bacteriophage, a luminescence-labelled bacteriophage or a Raman-labelled bacteriophage, (ii) a second chamber, and (iii) a membrane, which is arranged between the first chamber and the second chamber, wherein the membrane is impermeable to the bacteria to be determined and is permeable to labelling reagent that is not bound to the bacteria to be determined.

11. The measuring cell according to claim 10, wherein the volume of the first chamber is 2 to 20 ml.

12. A device for quantitatively determining bacteria, comprising: (i) a measuring cell according to claim 10, (ii) a means for withdrawing any excess labelling reagent from the first chamber and passing it into the second chamber, (iii) a means for exciting radiation, preferably fluorescence radiation, luminescence radiation and/or Raman radiation of labelled bacteria that are collected on the membrane, and (iv) a means for quantitatively evaluating a measurement signal originating from the bacteria collected.

13. A method for quantitatively determining bacteria in a sample, comprising the steps of: (a) incubating the sample to be tested together with a labelling reagent that can bind to the bacteria to be determined in the measuring cell according to claim 10, wherein the labelling reagent is a labelled bacteriophage, and is used in excess with respect to the bacteria to be detected, (b) removing labelling reagent that has not bound to the bacteria from the bacteria by means of the membrane that is impermeable to the bacteria to be determined and is permeable to the labelling reagent, wherein the bacteria to be determined are collected on the membrane, (c) detecting the labelling reagent bound to the bacteria collected, preferably by means of fluorescence measurement, luminescence measurement and/or Raman measurement, and (d) quantitatively evaluating a measurement signal from step (c).

14. The method according to claim 13, wherein said labelling reagent is selected from the group consisting of a fluorescence-labelled bacteriophage, a luminescence-labelled bacteriophage and a Raman-labelled bacteriophage,

15. The method according to claim 13, wherein said quantitatively determined bacteria is Legionella in a water sample.

16. A method for quantitatively determining bacteria in a sample, comprising the steps of: (a) incubating the sample to be tested together with a labelling reagent that can bind to the bacteria to be determined in the device according to claim 10, wherein the labelling reagent is a labelled bacteriophage, and is used in excess with respect to the bacteria to be detected, (b) removing any labelling reagent that has not bound to the bacteria from the bacteria by means of the membrane that is impermeable to the bacteria to be determined and is permeable to the labelling reagent, wherein the bacteria to be determined are collected on the membrane, (c) detecting the labelling reagent bound to the bacteria collected, preferably by means of fluorescence measurement, luminescence measurement and/or Raman measurement, and (d) quantitatively evaluating the measurement signal from step (c).

17. The method according to claim 16, wherein said labelling reagent is selected from the group consisting of a fluorescence-labelled bacteriophage, a luminescence-labelled bacteriophage and a Raman-labelled bacteriophage,

18. The method according to claim 16, wherein said quantitatively determined bacteria is Legionella in a water sample.

Description

[0052] The invention is also intended to be explained using the following figures:

[0053] FIG. 1 shows a measuring cell, which is formed as a measuring cuvette. The measuring cell contains a reaction chamber or first chamber (10) for receiving a sample volume of for example 10 ml. A label store (12) having a predetermined amount of labelling reagent, preferably in dry form, is provided in the first chamber (10). The first chamber (10) contains an opening (14) on one side for receiving the sample. Furthermore, the first chamber (10) is connected to a second chamber (18) by means of a membrane (16). The membrane (16) is designed so as to be impermeable to bacteria present in the first chamber (10) but to be permeable to labelling reagent that is not bound to bacteria.

[0054] For the measurement, the liquid in the first chamber (10) is passed into the second chamber (18) following incubation together with the labelling reagent. Bacteria that are present in the sample and comprise labelling reagent bound thereto are deposited on the membrane (16). Labelling reagent that is not bound can pass through the membrane (16) and enters the second chamber (18), together with the liquid. The bacteria deposited on the membrane (16) can be measured in situ, without additional measures.

[0055] FIG. 2 shows the result of a spectral measurement on a sample that has tested positive for the bacteria to be detected, for example E. coli or Legionella. The measurement signal is recorded by a spectrometer, the measuring range of which is preferably between 350 and 550 nm. The optical resolution of the spectrometer is preferably from 0.4 to 0.1 nm. The fluorescence signal originating from the bacteria to be determined is in the range of from 510 to 520 nm (ROI). A reference laser is used to ensure that the amplitude of the spectrum has a correct reference value. The evaluation comprises establishing two threshold values for bacterial counts of 100 CFU per 100 ml and 10,000 CFU per 100 ml. In the figure, the intensity of the measuring signal exceeds the threshold value of 10,000 CFU. Thus, the sample determined is extremely contaminated.

[0056] FIG. 3 shows the result of determining a sample with E. coli bacteria. E. coli phages coupled to the inorganic dye K.sub.2WO.sub.4:Eu (particle size of 5 to 15 nm) were used as the labelling reagent.

[0057] After incubation together with the labelling reagent in a reaction chamber, the bacteria contained in the sample were deposited on a membrane where they were determined. For this purpose, the membrane was homogeneously illuminated by a laser (405 nm/25 mW) and the emission radiation generated at a wavelength of from 610 to 615 nm was quantitatively determined. The quantitative conversion of the signal measured in corrected impulses into the number of colony-forming units (CFU) in the sample was possible by means of previous calibration using standard samples having a known number of E. coli bacteria, for example in the range between 10 and 10.sup.9 cells.

[0058] The peak of a Ca reference laser, which has been irradiated with an intensity corresponding to 10% of the threshold value of 10,000 CFU, can be seen at 702 nm in the spectrum.

[0059] Another signal, which is not used for quantitatively determining the bacteria, from the wolfram label contained in the dye can be detected in the range between 590 and 600 nm.

[0060] The determination of the sample shown in FIG. 3 resulted in a signal that corresponds to a bacterial count of 9,030 CFU. This bacterial count could be verified by using FACS for verification.