Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2001-02-08
2002-06-11
Gitomer, Ralph (Department: 2862)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S029000, C435S004000, C422S082010, C422S082020, C422S050000
Reexamination Certificate
active
06403333
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for determining whether microorganisms have retained biological activity.
The strength of the leakage current generated by the electrical field applied to a microorganism suspension in a dielectric oil gives information on whether or not biological activity is present. When followed by a microorganism collection and counting stage, the method can be used to determine the viability of the microorganisms after an electrical field has been applied.
Simple and rapid determination of the biological activity of microorganisms is essential in many fields such as pollution control, quality assurance, and clinical analysis. In research and in the food and agricultural industry, microorganisms such as bacteria and yeast are tools in increasingly widespread use. The use and effectiveness of the various biochemical processes involved depend on the viability of these microorganisms.
This being the goal, the standard technique consists of culturing a sample under specific conditions in an appropriate medium of the agar—agar type. If the items sampled and observed visually or under the microscope and counted are shown actually to reproduce, it may be determined that they are biologically active. In this case, the original strain is declared to be living. If not, it is concluded to be biologically inactive.
This method has a number of drawbacks, particularly the time required for the result to be obtained. In the best case, 18 hours are needed for visible colonies to form.
2. Description of the Related Field
EP Patent 0543090 proposes an analytical method based on direct measurement of the metabolic activity of bacteria, using a bioelectrochemical cell. Several aspects of the bioelectrochemical detection system are described in EP Patent 0221663 relating to a method and an instrument for measuring microbial activity in a bioelectrochemical cell, EP 0219247 relating to the bioelectrochemical cell and its electrodes, EP 0238322 relating to bioelectrochemical mediators, and EP 0352138 relating to improved electrodes used in bioelectrochemical cells. Some of these methods involve a step prior to analysis in which the bacteria are concentrated on a filter, an essential element of the device. Others use an electrode with a special configuration and special composition, made of expensive precious metals or porous carbon.
Whatever the systems described in these patents, the use of a chemical mediator is essential. This mediator is an organic compound such as 1,4-benzoquinone, which is dissolved in the medium and serves for transferring electrons between the electrodes and the microorganisms. When it contacts bacteria, it generates a response which is detected and measured by the measuring electrodes. The responses measured vary greatly depending on the mediator, its concentration, and its degree of oxidation, making it difficult to interpret and compare the results. Moreover, if any contaminants or elements able to exchange electrons with the mediator are present, numerous artefacts result. Finally, the presence of the mediator and a filter make it difficult to recover the microorganisms studied at a later time.
When a bioelectrochemical cell is used, the microorganisms are subjected to electrical fields in an aqueous medium, particularly in a buffer at the specified ionic concentrations. The high-frequency alternating currents produce alternating or rotating electrical fields. Such conditions do not allow voltages higher than a few volts to be applied for more than a few seconds. When direct current is used, the electrical voltage produced by capacitor discharge is always transient or pulsed. It can be kept at a high value, several kilovolts, for only very short times, less than one second, so that discontinuous or stepped measurements cannot be performed.
SUMMARY OF THE INVENTION
The goal of the invention is to overcome the above drawbacks by offering a rapid method for determining the state of a microorganism culture, adaptable to various microorganism species.
Another goal of the present invention is to furnish a device designed for simple and inexpensive implementation of the above method. Another goal is to enable the microorganisms studied to be recovered once an electrical field according to the method of the invention has been applied.
For this purpose, the invention relates to a method for determining the existence of microorganism biological activity by applying an electrical field, characterized by including application of an electrical field to a dielectric medium comprised of microorganisms suspended in a dielectric oil and measuring a characteristic leakage current when the microorganisms are alive.
For making the cell suspension, the microorganisms are first removed from the surface of an agar culture medium then placed in a dielectric oil. The dielectric oil can be a mixture of oils or a mineral or organic oil, preferably a silicone oil. The microorganism/dielectric oil mixture can be homogenized with an ultrasound probe.
The method according to the invention consists of gradually applying an electrical field to the dielectric suspension from the value zero to the maximum selected value. Application of the electrical field creates a leakage current in the medium containing the microorganisms and the dielectric oil. When the microorganisms are alive, the variations in leakage current strength as a function of the electrical field applied are characteristic and indicate whether biological activity is present. Whatever the stress resulting from application of the electrical field, a peak characteristic of the viability of the microorganisms studied appears. When the microorganisms are dead, on the other hand, the change in strength observed is a constant function of the electrical field appied.
The invention also relates to a device for determining whether microorganisms are biologically active, characterized by comprising a container equipped with electrodes in which the microorganism suspension is placed in a dielectric oil, means for generating an electrical field between two or more electrodes, measuring means, and means for recording the leakage current induced. The container in which the microorganism suspension is introduced is equipped with metal electrodes.
According to another embodiment of the invention, the container in which said suspension is introduced is comprised of an insulating plate provided with an inter-electrode space. Preferably, the voltage of the electrical current between the electrodes varies from 0 to 250 volts per millimeter.
Application of the electrical field, according to the method of the invention, may be followed by transfer to an aqueous medium, followed by counting. This application of the method is a simple and effective means of selecting the microorganisms that survive the electrical field.
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Hamilton, W. A. et al., “Effects of High Electric Fields on Microorganisms. II. Mechanism of Action of the Lethal Effect,” Biochimica et Biophysica Acta, vol. 148, pp. 789-800, 1967.
Anglade Jean-Yves
Barsotti Nathalie
Biosrayon Michel
Denunzio Dejugnac Corinne
Le Petit Jean
Chaudhry Mahreen
Etat Francais represente parlle Delegue General pour l'Arme
Gitomer Ralph
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