Filtration method for detecting microbial contamination

Abstract

Disclosed is a convenient sample preparation method for a medium suspected of containing contaminants, the method comprising a) passing a known volume of said medium through a filter from an influent side to an effluent side thereby concentrating the contaminants on the influent side of the filter, b) contacting the influent side of the filter with a liquid vehicle containing at least one substrate that through interaction with the contaminants each produces a detectable moiety, c) and allowing the substrate to interact with the contaminants on the influent side of the filter for a period of time, which is sufficient to allow the detectable moiety to be detected in the liquid vehicle. The method may further comprise a detection step, where the amount of detectable is determined in the liquid vehicle, preferably after the liquid vehicle has been separated from the contaminant, e.g. by passing the liquid vehicle through the filter and performing a measurement on the contaminant free liquid vehicle. Also disclosed is a kit for exercising the inventive method.

1 . A sample preparation method for a medium suspected of containing contaminants, the method comprising a) passing a known volume of said medium through a filter from an influent side to an effluent side in a filter device thereby concentrating the contaminants on the influent side of the filter in the filter device, b) contacting the influent side of the filter in the filter device with a liquid vehicle containing at least one substrate such that through interaction with the contaminants each produces a detectable moiety, c) and allowing the at least one substrate to interact with the contaminants on the influent side of the filter in the filter device for a period of time, which is sufficient to allow the detectable moiety to be detected in the liquid vehicle. 
     
     
         2 . The method according to  claim 1 , wherein, prior to step a, the medium is passed through a prefilter that does not retain the contaminants, but retains larger particles. 
     
     
         3 . The method according to  claim 1 , wherein the contaminants are selected from the group consisting of bacteria; fungi, such as filamentous fungi and yeast; algeae; protozoans; spores from bacteria; fungal spores; and pollen, and fragments thereof. 
     
     
         4 . The method according to  claim 1 , wherein the medium is a liquid medium. 
     
     
         5 . The method according to  claim 4 , wherein the liquid medium is selected from the group consisting of environmental water, drinking water, hot water, industrial water, process water, cleaning in place water, a liquid extract of a solid material, a suspended or solubilised surface sample, and liquid industrial products such as cosmetics, pharmaceuticals, and foodstuffs. 
     
     
         6 . The method according to  claim 4 , wherein the viscosity of the liquid medium is reduced prior to step a. 
     
     
         7 . The method according to  claim 6 , wherein viscosity is reduced by means of dilution or by means of treatment with a chemical agent such as a solubility enhancing agent or a detergent. 
     
     
         8 . The method according to  claim 1 , wherein the medium is a gaseous medium. 
     
     
         9 . The method according to  claim 8 , wherein the gaseous medium is air, such as air from a sterile facility, a laminar air-flow device or environmental air. 
     
     
         10 . The method according to  claim 1 , wherein the filter has a pore size small enough so as to retain substantially all contaminants in the medium. 
     
     
         11 . The method according to  claim 10 , wherein the filter has a pore size large enough to let the detectable moiety pass through the filter. 
     
     
         12 . The method according to  claim 11 , wherein the pore size is at most 20 μm. 
     
     
         13 . The method according to  claim 11 , wherein the pore size is at least 0.1 μm. 
     
     
         14 . The method according to  claim 1 , wherein the at least one substrate produces the detectable moiety by being cleaved by an enzyme that is characteristic for the contaminants. 
     
     
         15 . The method according to  claim 14 , wherein the enzyme is selected from the group consisting of carbohydrases, proteases, lipases, esterases, amidases, sulfatases, nucleases and phosphatases such as alkaline phosphatase. 
     
     
         16 . The method according to  claim 14 , wherein the enzyme is expressed constitutively by microorganisms. 
     
     
         17 . The method according to  claim 14 , wherein the at least one substrate is a fluorogenic or chromogenic substrate producing blue, green and red fluorescent products as the detectable moiety. 
     
     
         18 . The method according to  claim 14 , wherein the at least one substrate is selected from the group consisting of 5-bromo-4-chloro-3-indolyl phosphate disodium salt; 9h-(1,3-dichloro-9,9-dimethylacridine-2-one-7-1) phosphate ammonium salt; fluorescein diphosphate tetraamonium salt; a methylumbelliferyl derivative such as 6,8-difluoro-4-methylumbelliferyl phosphate, 4-methylumbelliferyl phosphate dicyclohexylammonium salt trihydrate, 4-methylumbelliferyl phosphate free acid; 4-methylumbelliferyl phosphate dilithium salt, 4-methylumbelliferyl-PNacetylglucosaminide, and trifluoromethylumbelliferyl phosphate; salts of 4-nitrophenyl phosphate; and resorufin phosphate. 
     
     
         19 . (canceled) 
     
     
         20 . The method according to  claim 1 , wherein at least two substrates are used that produce detectable moieties providing signals that can be combined into one single measured signal value. 
     
     
         21 . The method according to  claim 1 , wherein at least two substrates are used that produce detectable moieties providing distinguishable signals. 
     
     
         22 . The method according to  claim 1 , wherein the contaminants are viable microorganisms. 
     
     
         23 . The method according to  claim 1 , wherein the amount of substrate in the liquid vehicle does not limit the rate of production of the detectable moiety. 
     
     
         24 . The method according to  claim 23 , wherein the rate of production of the detectable moiety is a function of the quantity of contaminants in the known volume of the medium. 
     
     
         25 . The method according to  claim 24 , wherein the function is linear. 
     
     
         26 . The method according to  claim 1 , wherein several different known volumes of the medium are each passed through a filter in step a, so as to ensure that at least one of the volumes contains a suitable number of contaminants. 
     
     
         27 . The method according to  claim 1 , wherein the filter is part of a closed, sterile filter device. 
     
     
         28 . The method according to  claim 27 , wherein the closed, sterile filter device is disposable. 
     
     
         29 . The method according to  claim 27 , wherein the closed, sterile filter device integrates the filter and a filter housing into one irreversibly closed structural unit. 
     
     
         30 . The method according to  claim 28 , wherein the longest cross-sectional axis of the closed, sterile filter device does not exceed a length of 10 cm. 
     
     
         31 . The method according to  claim 1 , wherein the interaction in step c is terminated by interrupting the contact between the at least one substrate and the contaminants. 
     
     
         32 . The method according to  claim 31 , wherein interruption is obtained by evacuating the liquid vehicle from the filter device while retaining the contaminants in the filter device. 
     
     
         33 . The method according to  claim 32 , wherein the liquid vehicle is evacuated from the filter device in the direction from the influent to the effluent side of the filter. 
     
     
         34 . The method according to  claim 33 , wherein evacuation is obtained by applying an elevated pressure on the influent side of the filter or by applying a lowered pressure on the effluent side of the filter. 
     
     
         35 . The method according to  claim 1 , wherein the interaction in step c is terminated on the filter. 
     
     
         36 .- 47 . (canceled)