Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2002-05-24
2004-08-17
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S027000, C435S029000, C435S004000
Reexamination Certificate
active
06777201
ABSTRACT:
The present invention concerns a method for the determination of the amount of micro-organisms and/or biological activity in a sample. The invention primarily concerns the determination of the amount of aerobic micro-organisms in a fluid sample taken from a process fluid
BACKGROUND OF THE INVENTION
Micro-organisms are present practically everywhere and they exhibit an extraordinary power to adapt even to very hostile environments. It is important to be able to determine the amount of micro-organisms in process fluids and other solutions, e.g. in order to adjust the amount of process chemicals, to ensure that set limits are not exceeded, and in order to monitor processes. It is also important to be able to monitor the amount of micro-organisms, in order to quickly respond to changes. As examples of process fluids can be mentioned paper pulp and process fluids associated therewith, product flows and wash- and rinse liquids in food industry, municipal and industrial waste waters, fluids within other process industry, such as textile industry, water reservoirs, swimming pools etc.
The most frequently used method for determination of the concentration of micro-organisms and/or microbiological activity in fluids is based on counting the number of micro-organisms in a sample. Several methods are in use, of which the large majority are based on cultivating the micro-organisms in or on a medium, e.g. on agar plates or so called dipsticks. The cultivation requires a suitable environment and an incubation time between one to five days. The result is obtained after a manual or automated count of the number of visible colonies. It has however been shown, that in some cases more than 90% of the micro-organisms, originally present in the sample, die either at the time of sampling, during transport or during the incubation which favours certain strains. The reliability of these methods should therefor be questioned, both when it comes to the number and the type of micro-organisms present in the sample.
Another approach is to study a sample under microscope and count the number of micro-organisms. This also allows identification of different species, but this is work-intensive and requires good knowledge of both the method and micro-organisms. This method is probably the most reliable in clean nutrient solutions, but in process solutions which often consist of suspensions of particles, e.g. fibres, flocs or other finely dispersed material, practical difficulties arise. One problem is that dead micro-organisms have to be separated from living ones, something which often is performed with the aid of some colouring procedure. Another problem is that the micro-organisms often are attached to the particles, e.g. to the fibres in a fibre suspension. Therefor, a study of a filtrate does not give a fair picture of the amount of micro-organisms in the real process solution. For these reasons, among others, a determination using microscopy would only exceptionally be used for routine examination of process solutions.
Prior art methods for the determination of the amount or concentration of micro-organisms are accordingly associated with many problems, e.g. insufficient accuracy and reliability due to the micro-organisms dying at the time of sampling, transport and/or incubation of the samples; errors due to the sample not being representative for the process solution (e.g. a filtrate); requirements that the determination is performed by skilled personnel; work-intensive procedures; time-consuming incubations.
The present invention sets out to solve these problems, among others by making available a method which is rapid, suitable for automatisation, independent of the constitution of the process solution, requires minimal work effort and maintenance of the equipment. Further benefits of the invention will become apparent from the description and example.
CLOSEST PRIOR ART
The use of catalase activity as a measure of microbiological activity in different applications within the pulp and paper industry is Known (D. Y. Prasad, Tappi Jounal, January 1989, 135-137). The disclosed measurements where however done on filtrates and compared to conventional incubation of agar plates. Knowing the amount of micro-organisms removed by filtration and the unpredictability of incubation methods, the accuracy of the report's conclusions should be questioned.
A widely held point of view (see e.g. Y. Vemac et al. Progress in Paper Recycling, February 1999, 83-88) seems to be that the relation between the amount of micro-organisms and the catalase concentration is uncertain and varying, i.e. due to the availability of nutrients, self poisoning and death among the micro-organisms, and the variations in microbial flora between different plants for the production of pulp and/or paper.
A method for the detection of micro-organisms through the measurement of catalase activity is disclosed in WO 94/03632. The micro-organisms are allowed to convert a substrate, hydrogen peroxide, to oxygen and water over a controlled period of time under buffered conditions. At the end of this controlled period of time, the remaining substrate is determined eletrochemically by the peroxidase catalysed rupture of carbon-fluorine bonds from an organo-fluoro compound using a fluoride ion sensitive electrode.
There remains a need for a reliable, rapid and simple method for online determinations of the amount of living micro-organisms in process solutions, and in particular in process solutions with suspended particles or fibres. Prior art methods are not only less suited for use with process solutions, but also fail in distinguishing between living and dead micro-organisms.
SUMMARY OF THE INVENTION
The present invention aims primarily to make available a method which makes it possible to determine the amount and/or concentration of aerobic micro-organisms in any process solution, rapidly and reliably, and with reduced requirements with regard to the knowledge and work effort necessary from the part of the user. This is achieved through a method according to the attached claims, which method simultaneously solves the problems associated with the prior art methods.
According to the inventive method, the concentration of a substance secreted by micro-organisms, is determined in a sample, and the micro-organisms killed, whereupon the concentration of said substance is measured anew, and the relation between the first measured concentration and the second concentration is used a measure of the amount of living micro-organisms originally present in the sample.
REFERENCES:
patent: 53-127880 (1978-11-01), None
patent: 57-074095 (1982-05-01), None
patent: 50-15396 (1993-01-01), None
patent: WO 94/03632 (1994-02-01), None
Shepherd et al, (Antimicrob. Agents Chemother; V.32(11), p.1693-1698(1998)Abstract Only.
Bergsland Helena
Fladda Gerdt
Norder Stig
BTG Kalle Inventing AB
Leary Louise N.
Young & Thompson
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