Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2000-05-26
2004-04-20
Redding, David A. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C422S082050, C356S300000, C356S317000, C356S318000, C356S337000, C356S432000, C356S445000, C356S451000
Reexamination Certificate
active
06723554
ABSTRACT:
The present invention relates to an apparatus and a method for measuring the optical properties of a medium, and a corresponding micro-bioreactor. These optical properties may in particular include the turbidity, the absorbance and the fluorescence of the medium, at one or more wavelengths or over predetermined spectral ranges.
In many biological laboratories and also in certain industries, it is very often necessary to cultivate micro-organisms in liquid medium and to measure the concentration of the cells at different stages of the culture. This quantification may be performed directly on samples, by counting the cells or by measurement of the dry weight. These methods, although very accurate, are long and tedious, and it is more convenient to evaluate the quantity of the cells indirectly, for example by measuring the disappearance of substrates or the appearance of intracellular (NADH, etc.), or extracellular compounds (alcohols, organic acids, etc.). However, the most convenient and most frequently used methods for evaluating the cell concentrations of cultures are based on their optical properties.
When matter is struck by electromagnetic radiation, this interacts with the electronic charges of the atoms: part of the radiation passes through the matter without change of direction (transmitted radiation) and part is diffused in all directions, each illuminated particle then behaving as a light source. The quantity of light diffused by the particles present in the liquid medium increases with the number and size of these particles; in addition, in the case where the particles are micro-organisms, it has been shown that the diffusion of the light does not occur homogeneously in all directions, but predominantly in a direction close to that of the incident beam.
It is these diffusion phenomena which cause liquid micro-organism cultures to have a cloudy appearance, whose density increases with the cell concentration. Devices which can quantify this cloudiness, or turbidimeters, measure the turbidity of these cultures.
Existing turbidimeters may be divided into discontinuous measurement devices, which are used to make repeated measurements of the light transmitted and/or reflected by a liquid medium, and on-line measurement devices, for which a turbidity measurement probe is inserted into the liquid medium and connected to a recording system.
Discontinuous measurement devices, such as nephelometers, colorimeters, spectrophotometers and mixed turbidimeters, cannot for the most part measure high turbidity levels. Their measurement range does not normally extend beyond 1000 NTU (Nephelometric Turbidity Units) and is most often much more limited. It is thus necessary to dilute the samples, which increases the risks of error and the work to be performed by the operator. In addition, the sampling itself is very restricting, since it requires the presence of the operator at regular intervals throughout the culture period. Some mixed turbidimeters have been designed to cover a wider turbidity range, but they are costly.
On-line turbidimeters, whose measurement range may be fairly wide, have the disadvantage of requiring large culture volumes (about 1 litre) because of the size of the probes introduced into the cultures, which limits their use within bioreactors. In addition, cultures in bioreactors are generally aerated and stirred, which generates numerous bubbles which interfere strongly with turbidity measurement. These devices are also costly.
International patent application WO-92/13.482 discloses an apparatus and a method for measuring a blood parameter. The apparatus comprises a red light source and an infrared light source directing the light towards a blood sample and a detector receiving the light generated by the two sources and reflected by the blood. An optical feedback loop is used for each of the two sources so that the intensity of the light received by the detector is approximately constant for a range of values of the blood parameter, which allows precise control of the light sources. Reference curves are used to calculate two blood parameters from the feedback signals obtained respectively for each of the two sources.
A disadvantage of this technique is that it is in particular limited by the transmission capacities of the light sources and thus only covers a restricted measurement range with sufficient sensitivity. In addition, obtaining each result requires a stabilisation time which prevents rapid measurement. A serial analysis of a large number of samples, or measurement in heterogeneous media and /or media containing gas bubbles thus proves very difficult, or even impossible. In addition, inaccuracies can be generated during the processing of the feedback signals, because of the drift of capacitance values.
U.S. Pat. No. 4.447.150 relates to a device and a method for measuring blood properties and parameters, used to measure the oxygen saturation level of the blood. The apparatus disclosed comprises two light sources transmitting respectively at two distinct wavelengths and a detector measuring the light generated by the two sources and reflected by the blood. The light reflected or transmitted by the blood is maintained at a constant level by means of an optical feedback loop. In addition, the ratio of the two voltages corresponding to the measured light levels from the two sources respectively gives the percentage of saturation of the blood oxygen.
This technique also has the disadvantage of only allowing a restricted measurement range, in addition to the disadvantages already mentioned for document WO-92/13.482.
The present invention provides a measurement apparatus and a method which do not have the above disadvantages, and thus allow measurements over different ranges of values while retaining good sensitivity.
The apparatus and method of the invention can also offer a high speed of measurement and good accuracy.
In particular, the object of the invention is an apparatus for measuring turbidity able accurately to measure the turbidity of microbial cultures over a wide range of values, which is reliable, easy to use, readily adaptable to micro-bioreactors, of small size and inexpensive.
A further object of the invention is an apparatus for measuring absorbance and an apparatus for measuring fluorescence, able to measure with the same advantages as cited above the molecular absorbance and/or fluorescence of a medium, and, more generally, the invention provides an apparatus for measuring one or more optical properties of a liquid or solid medium.
The invention relates to a measurement apparatus as described above which is advantageously automated, so that experiments may be performed over a long time period without the presence of an operator.
Another object of the invention is a measurement apparatus as described above suitable for a battery of micro-bioreactors, making simultaneous measurements for the different micro-bioreactors possible.
The invention also relates to a micro-bioreactor equipped with a measurement apparatus having the above properties.
A particular object of the invention is a micro-bioreactor having systems for supplying the culture medium and gas which are simple and reliable, protected from any external contamination and able to handle small culture volumes. The system similarly protects the external environment from contamination by the culture itself.
The invention also relates to a micro-bioreactor with automatic sampling at certain predetermined stages of turbidity, absorbance, and/or fluorescence, and a micro-bioreactor with automatic dilution for cyclic cultures.
A further object of the invention is a method for measuring optical properties which may include in particular turbidity, absorbance and fluorescence, making possible measurement over a wide range of values with good accuracy, reliability and reproducibility.
The invention may be applied in the fields of microbiology and biotechnology, and more generally for any measurement of turbid substances, for example for analysis and treatment of water, agricultural or industrial e
Gaillon Laurent
Longin Robert
Luu Thanh Dung
Institut Pasteur
Redding David A.
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