Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-08-09
2003-05-13
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S023000, C435S836000, C435S968000
Reexamination Certificate
active
06562583
ABSTRACT:
BACKGROUND OF THE INVENTION
In certain spaces with special requirements for the state of the air, such as in air-conditioned rooms, clean rooms and isolation areas, the air is regularly analyzed as to its microbial content. Since filtered air generally is involved, which tends to exhibit a low microbial content, large volumes are usually tested in order to collect sufficient microorganisms for a representative test result. To that end an air sample is typically collected by an air collection apparatus, such as is marketed by the firm Sartorius under the name MD8 air scan and filtered through an appropriate filter. Filters employed for this purpose are sterile membrane filters with pore sizes as prescribed in microfiltration technology, predominately of gelatin. See, for example, DE PS 11 73 640. Such gelatin membrane filters retain microorganisms and hold them in a moist and reproductive condition. Following the sampling, the gelatin membrane filters can be incubated on an agar culture medium whereby, from the individually collected microorganism aggregates, colonies are grown; the gelatin filter liquefies and disappears so as to permit the microorganism colonies to be counted directly on the agar. In another alternate method, the gelatin filter can be dissolved in a sterile solution, such as a peptone solution of water or an isotonic salt solution, so that representative quantities can be incubated on various culture media. In any case, in accord with these procedures, the analysis results are received long after the fact, since the typical microbial colony growth rate is on the order of seven days.
A much more rapid microbiological assay, known as ChemScan® is theoretically available for use with such gelatin-based filters, allowing a representative microbial count within 30 to 90 minutes. For this type of analysis an aqueous sample containing the living microorganisms would be filtered through a 0.22 &mgr;m or a 0.45 &mgr;m analysis membrane in order to retain the microorganisms captured by the gelatin membrane filter. The analysis membrane would then be laid upon an absorption substrate for 30 minutes at 30° C. and saturated with an enzymatic marking fluid. The marking fluid is capable of causing enzyme-activated interaction with the microorganisms' cell cytoplasm, ideally yielding a yellow fluorescence. Finally, by means of a microscope, preferably with a laser scan, individual microorganisms on the analysis membrane should be detectable by virtue of their fluorescence. Just as in the case of conventional assays, such a rapid microbiological assay may convert the microorganism-containing gelatin filter to a test sample by dissolving the gelatin filter in an aqueous solution such as an aqueous peptone solution. Unfortunately, attempts to conduct such a rapid assay conducted with conventional gelatin membranes have not yielded an observable yellow fluorescence on the analysis membrane as the analysis membrane remains red-colored throughout the procedure.
Thus the invention has the object of providing a procedure for a rapid assay of microorganisms and to that end the related object of providing suitable gelatin membrane filters for use in such rapid assays. These objects and others which will become apparent to one of ordinary skill in the art are summarized and described in detail below.
BRIEF SUMMARY OF THE INVENTION
The essence of the present invention lies in the discovery that gelatin membranes that are free from microscopic particles greater than 0.45 &mgr;m in diameter provide vastly superior microorganism-collecting membrane filters for purposes of utilizing the latest rapid microbiological assay method noted above.
The invention also provides a method for rapid and near real time determination of microorganisms in gases, comprising the following steps:
(a) providing a gelatin membrane filter free of particles greater than 0.45 &mgr;m in diameter;
(b) forming a membrane containing microorganisms by contacting the membrane of step (a) with a gas containing microorganisms;
(c) forming a solution containing the membrane-captured microorganisms by dissolving the membrane containing microorganisms of step (b) in an aqueous solution containing an enzymatic marking substance which causes the microorganisms to fluoresce;
(d) collecting flourescent microorganisms by filtering the solution of step (c) through an analysis membrane having a pore size of from about 0.2 to about 0.45 &mgr;m; and
(e) counting the fluorescent microorganisms of step (d).
The method can be used for the determination of microorganisms in gaseous media, especially air, in pharmaceutical, biotechnical, and food industries. Further, application is found in environmental operations, in waste industries, and in medical apparatus for the determination of germ contamination of the gaseous media. The gelatin membrane filters, in accord with the invention, may be used in combination with a collection device for the capture of bacteria, spores, viruses, yeasts and microorganisms to determine the degree of germ contamination in a given space.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
According to the present invention there are provided gelatin membranes that are free from microorganisms, including dead microorganisms. Rather surprisingly, it has been discovered that the use of gelatin freed from microscopic particles as a starting material for the manufacture of a gelatin membrane filter provides a superior microorganism-capturing filter. To this end, a casting solution of commercial gelatin is first filtered under pressure through a microfiltration membrane having a pore size diameter of up to about 0.45 &mgr;m, preferably up to about 0.2 &mgr;m, and most preferably up to 0.1 &mgr;m. A hydrophilic, cross-linked, 0.2 &mgr;m cellulose hydrate membrane has proven itself as especially appropriate as such a microfiltration membrane, commercially available as Hydrosart®, from Sartorius AG of Gottingen, Germany. By such pre-filtration of the gelatin from which the membrane filters are cast, all particles of the stated size are removed from all the components of the membrane casting solution in a single step. Pressure filtration is preferable as compared to vacuum filtration, since volatile solvents cannot vaporize and the composition of the membrane casting solution is not altered.
A commercial gelatin membrane filter available from ChemScan® made and sterilized directly, without the inventive pre-filtration step can contain between about 10,000 and 1,000,000 dead microorganisms per gram. Such microorganism counts are obviously responsible for the failure of fluorescent marking of membrane filters in the rapid test procedures employed in the ChemScan® process inasmuch as microorganisms, especially dead microorganisms, cause a red coloration in the analysis membrane, thereby preventing detection of the fluorescence of living microorganisms.
The gelatin membrane filters in accord with the invention are well adapted in particular for rapid microbiological assays for the direct determination of individual microorganisms, in particular living microorganisms, which may be identified by fluorescence. The gelatin membrane filter upon which microorganisms have been captured is dissolved in an aqueous solution, filtered through an analysis membrane with pore sizes ranging from about 0.2 &mgr;m to 0.45 &mgr;m, in order to collect the microorganisms. For this step micro-sieve membranes are preferred, as described in WO 95/13860 A1, especially nuclear track etched membranes with pore diameters of about 0.2 &mgr;m, as they have a narrow pore size distribution and because of the abundance of pore openings perpendicular to the membrane surface, which permit high filtration rates. They are also preferred because microorganisms are retained completely on the surface of the membranes and not, as is the case with conventional membranes, inside the pore structure of the membrane. The analysis membrane may then be placed for, say, 30 minutes at 300° C. on an absorptive substrate and then impregnated with an enzymatic marking age
Herbig Elmar
Nussbaumer Dietmar
Vinh Khuong To
Chernoff , Vilhauer, McClung & Stenzel, LLC
Leary Louise N.
Sartorius AG
LandOfFree
Method for detecting microorganisms in gases does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for detecting microorganisms in gases, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for detecting microorganisms in gases will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3070832