Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2001-10-03
2003-09-30
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S029000, C435S030000, C435S031000
Reexamination Certificate
active
06627413
ABSTRACT:
DESCRIPTION
1. Technical Field
The present invention relates to a process for quantitative and/or qualitative determination of the microbial contamination of aqueous suspensions, emulsions or dispersions containing minerals and/or pigments and/or fillers and/or fiber materials, optionally in combination with polymers in colloidal form.
2. Related Art
Determinations of the germ contamination and hygienic controls of aqueous suspensions by means of conventional methods essentially rely on the propagative capacity of the microorganisms to be detected. Naturally, the time required to perform these methods ranges from 24 h to several days. These time periods are too long for many questions, and the results are obtained too late to intervene in a guiding manner in production processes. Particularly performing controls prior to transport time and again requires methods which are characterized by short determination intervals.
Thus, the main problem of conventional microbiological analyses of aerobic mesophilic germs, such as Plate Count or Easicult, is their long incubation period of up to 48 h. By this methods it is impossible to obtain an evaluation and, thereby, determination of the germ count earlier than after the elapse of two days. In addition, several other effects must be considered such as the nutrient medium, partial pressure of oxygen (aerobic/anaerobic), selectivity, pH and much more.
Therefore, it is often impossible to determine the germ count of products, such as pigment slurries, prior to their shipment to customers and to intervene in a guiding manner by adding biocidal substances. Due to the long transport times required of up to 6 weeks by deep sea ship or rail, the pigment slurry may deteriorate or become unusable. The white pigment slurry may develop a gray color and start to smell. To date, preventive over-dosage of biocidal substances in the pigment slurry is the only possibility to exclude spoilage, is very cost-intensive, dissipates resources, and is ecologically nonsensical. Moreover, two different approaches are necessary to analyze for both aerobic mesophilic germs and fungi. Furthermore, the preparation of serial dilutions is necessary, thus multiplying the number of analyses.
Conventional methods for the determination of germs in the paper and pigment industries have been for example described in the “Schweizerisches Lebensmittelbuch”, chapter 56, section 7.01, 1985 edition, 1988 revised version, “Bestimmung von aeroben Bakterien und Keimen”, and in the “Schweizerisches Lebensmittelbuch”, chapter 56, section 7.22, 1985 edition, 1988 revised version, “Bestimmung von Pilzen”. Generally, prior to performing a determination in each case an incubation period of about 48 hours is required.
The CellFacts® particle analyzer and method have been developed by Microbial Systems company, Ltd. More detailed information may be obtained from Labor flash 9/96, Zeitung mit Leserdienst für Labor und Forschung, Ott Verlag+Druck AG, Ch-3607 Thun, Switzerland. Thus, it has been mentioned that the CellFacts analyzer may also be used to carry out determinations of the germ count in calcium carbonate slurries. However, experiments performed in the applicant's laboratory revealed that a determination of this type in the manner described by the manufacturer is impossible or only inaccurate.
The principle of the measurement performed by the CellFacts analyzer is based on the measurement of bacteria, fungi, and yeasts in the form of particles in an electrical field wherein the number of particles is determined by an interval incubation of the samples and subsequent measurement of the increase in particle count and, in the case of exponential growth, by extrapolation to t
0
. This measurement principle is also effective in measuring a “low” number of “foreign particles” having the same or a similar size as bacteria, fungi, and yeasts. In the case of suspensions and emulsions containing a proportion of “foreign particles”, such as minerals, fillers and/or pigments, of >1% by wt. the number of inert “foreign particles” present having the same size as microorganisms, namely 0.5-20 &mgr;m, i.e. the blank value t
0
, is too high to enable the detection of a further increase of the germs by propagation in the incubator within a period of <10 hours. Thus, the CellFacts analyzer is unable to perform a sufficiently exact measurement, and the manufacturer requires a dilution of the starting liquid to ensure applicability.
At a blank value of 10
8
particles/ml, it is impossible to significantly detect an increase of 10
3
particles/ml by means of the CellFacts analyzer. However, the dilution required due to the presence of the “foreign particles” is too high so that the dilution of the reproductive organisms which of course are diluted by the same order of magnitude is no longer significant, and the result obtained is incorrect. Furthermore, “foreign particles” having a diameter of >20 &mgr;m may plug the measurement cell which has a diameter of only 30 &mgr;m. The “foreign particles” may for example be of mineral type, such as calcium carbonate, synthetic, organic, of polymeric type, such as polystyrene acrylate dispersions, or of natural, organic type, such as starch solutions or hemicelluloses and/or cellulose fibers, or a combination of the above particles as they are for example present in a paper mill cycle.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a quick and powerful, easily performed process for the quantitative and/or qualitative determination of the microbial contamination of aqueous suspensions, emulsions or dispersions containing minerals and/or pigments and/or fillers and/or fiber materials wherein said process avoids the above described disadvantages of the prior art.
According to the present invention, this object has been solved by a process according to the generic part of claim
1
characterized in that a sample of the suspensions, emulsions or dispersions, following the addition of one or more organic substances which can be degraded by microorganisms and optionally following a subsequent incubation, is centrifuged to separate the microorganisms from the minerals and/or fillers and/or pigments and/or fiber materials and the number and/or size and/or nature of the microorganisms in the aqueous supernatant phase is determined after one or more incubations.
Preferred embodiments of the present invention will become obvious from the dependent Claims as well as the following Specification and the Examples.
DETAILED DESCRIPTION OF THE INVENTION
Since several years, the quantitative and qualitative determination of microorganisms belongs to the prior art. However, the determination of microbial contamination is particularly difficult in cases where a high concentration of other solid particles such as minerals, pigments, fillers and/or fiber materials is present in the sample to be investigated. Because these “foreign particles” often have a size similar to microorganisms, generally, it is only possible to minimize the number of such “foreign particles” by carrying out high dilutions. However, concomitantly and unavoidably, these high dilution steps reduce the number of contaminating microorganisms, and a long incubation period is required to increase the number of microorganisms by means of propagation to an extent that enables safe detection.
Therefore, there has been a need to create a new process of the type mentioned in the beginning which provides reproducible and reliable results with respect to the number, size and/or type of the microorganisms in the sample to be investigated in a substantially shorter time.
Surprisingly and unexpectedly, it has been discovered that it is possible to separate the microorganisms from inert materials (“foreign particles”) by addition of degradable organic substances to the sample and performing a subsequent centrifugation. Usually, the microorganisms preferably adhere to the surface of the mineral, pigment, filler and/or fiber materials making it difficult to separate them from the s
Buri Matthias
Schwarzentruber Patrick
Jenkins & Wilson & Taylor, P.A.
Omya AG
Saucier Sandra E.
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