Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
1999-08-11
2001-05-15
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S283100
Reexamination Certificate
active
06232091
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to devices and methods for determining and/or analyzing properties of suspensions and solutions, and more particularly an apparatus and method for determining and analyzing the cell growth parameters in a liquid biological culture medium.
2. Description of the Related Art
Many industrial and laboratory processes require the periodic or continual measurements of the cell growth parameters such as cell concentration, cell size distribution and relative quantity of viable cell suspended in liquid or in culture medium. These data are critical to the proper control of variables which influence cell growth rate (e.g. temperature, pH, nutrient levels, etc.).
Modern advancements in the science of recombinant gene technology have given rise to new possibilities for large scale production of difficult to synthesize chemical substances, through the controlled fermentation or culture of genetically recombinant microorganisms, as well as “wild type” microorganisms. The industrial fermentation or culture of genetically recombinant microorganisms is typically effected by growing the recombinant microorganisms under aseptic conditions within a liquid growth medium contained in a bioreactor or fermenter vessel. Such bioreactor or fermentation vessels generally comprise an enclosed vessel outfitted for microprocessor or computer effected control of variables such as feed rate, temperature, pH and oxygen content. Periodic or continuous measurements of cell density by optical method are utilized as a basis for making corresponding changes in process variables. See, e.g., L. A. Tam, Method and Device for Measuring and Controlling Cell Density in Microbiological Culture, U.S. Pat. No. 5,483,080; W. R. Mandel, A. J. Dekovich, Apparatus and Method for Optical Density Measurements of Biomass Processes, U.S. Pat. No. 4,893,935. This method gives only the information about cell or product concentration in a biological reaction system.
Another apparatus was developed for directly monitoring microorganisms in a bioreactor by a TV camera that has an optical magnifying system and a refilled sample cell for cell suspension. By analyzing the cell images on a computer program, it is possible to receive the cell sizes and cell concentration in the culture medium (T. Saito, at. al., Microorganism Monitoring Apparatus, U.S. Pat. No. 4,661,845.). This method though is expensive and does not give information about content of viable cells in suspension.
Another approach to determinate the cell concentration and cell size distribution in cell suspension is used by simultaneous measuring an optical and electrical impedance on a stream of particles or cells passing through a particle sensing aperture. This method is known as flow cytometry. In all known devices the electrical field is used only for cell sorting. See, e.g., J. D. Hollinger, R. I. Pedroso, Parting Analyzing and Sorting Apparatus, U.S. Pat. No. 4,515,274; K. Yamamoto, et al., Cell Analyzer, U.S. Pat. No. 5,408,307.
To monitor the growth of microorganisms in liquid culture another method is used comprising the detection of pressure change in a gas-tight container incorporating a flexible diaphragm. See, e.g. N. T. Court, et al. Method and Apparatus for Monitoring the Growth of Microorganisms in Liquid Culture, U.S. Pat. No. 5,863,752.
An apparatus is known that comprises electrodes which can be introduced into a cell culture sample for measuring an electrical impedance of the suspension. The growth or multiplication of microorganisms is determined in terms of the impedance measurements. See, e.g. G. Franzi, Apparatus for the Automatic Monitoring of Microorganism Culture, U.S. Pat. No. 5,432,086; P. J. Malin et al. System for Electronically Monitoring and Recording Cell Culture, U.S. Pat. No. 5,643,742.
It has been proposed to detect the presence or absence of living microorganisms by treating the cell suspension special chemical markers of fluorochromes and monitoring light emission produced by cell suspension. Viable and nonviable cells have the different specificity for the markers and accordingly emit or do not emit the light. (J. R. Clendenning, Method and Detecting Living Microorganisms, U.S. Pat. No. 3,933,592; J. W. Steele, F. Sribnik, Automated Biluminescence Microbial Monitor, U.S. Pat. No. 5,141,869.) The classical method of determination of viable cell quantity in culture is early detection and enumeration of colonies in the growth medium (S. D. Morgan, Method for Rapid Quantification of Microorganism Growth, U.S. Pat. No. 5,510,246).
In general, it is known that when living cells are disposed in a liquid and the liquid is placed in alternating electrical field, the cells will tend to be oriented in the field, even if the dielectric properties of the cells and surrounding medium are isotropic. Due to the nonspherical shape of microbial cells, the mean potential energy of the system depends on the orientation of the cells with respect to the field. The direction corresponding to the minimum energy determines the stable state of orientation. For instance, for pure and isotropic dielectrics, it is well known that an ellipsoidal body always will be oriented with its longest axis parallel to the external field. Electrooptical Apparatus for Microbial Identification and Enumeration, described in U.S. Pat. No. 4,576,916 by G. E. Lovke and R. J. Meltzer, uses this cell orientation phenomena. The main destination of this device is the optical identification of microorganisms by low-frequency electrical field (<1 kHz). It should be noted that the high requirements for accuracy and stability of a electrooptical device by work with biological objects was not realized in a sufficient value in the known equipment.
SUMMARY OF THE INVENTION
The invention provides an electrooptical apparatus and method which takes advantage of the Kerr Effect to detect, monitor, measure and utilize the affects of electric fields.
The present invention describes an electrooptical apparatus and method for monitoring cell growth in microbiological culture. An apparatus performs the calculation in real-time of the cell growth parameters such as cell concentration, cell size distribution, and relative quantity of viable cell suspended in culture medium. For this purpose, a cell suspension sample is withdrawn automatically from a biological reactor or fermenter, diluted to specified optical density, and delivered into an electrooptical cell, having paired electrodes and two orthogonal optical channels for passing through two light beams. The present invention provides an electrooptical apparatus in which the optical density changes of suspension are measured in two orthogonal directions by inducing in electrooptical cell of an alternating electrical field in frequency range from 1000 Hz up to 100 MHz for determining an electrooptical cell response for a plurality of frequencies.
The present invention also provides an electrooptical apparatus in which the optical density changes can be simultaneously and precisely measured in two orthogonal directions for cell relaxation from partially oriented state to random state after turning off an electrical field.
In addition, the present invention provides an electrooptical apparatus in which an optimal measurement condition can be adjusted for each sample so as to increase the reliability of obtained results and to measure a large number of samples with high efficiency.
The invention will be more fully described by reference to the following drawings.
REFERENCES:
patent: 3933592 (1976-01-01), Clendenning
patent: 4250894 (1981-02-01), Frei et al.
patent: 4515274 (1985-05-01), Hollinger et al.
patent: 4576916 (1986-03-01), Lowke et al.
patent: 4661845 (1987-04-01), Saito et al.
patent: 4893935 (1990-01-01), Mandel et al.
patent: 5099848 (1992-03-01), Parker et al.
patent: 5141869 (1992-08-01), Steele et al.
patent: 5178148 (1993-01-01), Lacoste et al.
patent: 5265612 (1993-11-01), Sarvazyan et al.
patent: 5344535 (1994-09-01), Betts et al.
patent:
Artann Laboratories
Leary Louise N.
Mathews, Collins Shepherd & Gould P.A.
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