Method for recovering microbial cells

Abstract

The present invention provides a method of recovering viable microbial cells from a complex sample, said method comprising: a) providing a sample having a volume of at least 1 ml; b) contacting said sample with a buffer solution and one or more proteases, wherein said buffer solution has a pH of at least pH 6 and less than pH 11, wherein said buffer solution and said one more proteases do not comprise a detergent or a chaotrope, and wherein the buffer solution/protease/sample mixture is non-hypotonic; c) filtering the mixture obtained in step (b) through a filter suitable for retaining microbial cells; and d) recovering the microbial cells retained by the filter in step (c), wherein the recovered microbial cells are viable, and a microbial recovery device for the same.

1 . A method of recovering viable microbial cells from a complex sample, said method comprising:
 a) providing a sample having a volume of at least 1 ml;   b) contacting said sample with a buffer solution and one or more proteases, wherein said buffer solution has a pH of at least pH 6 and less than pH 11, wherein said buffer solution and said one more proteases do not comprise a detergent or a chaotrope, and wherein the buffer solution/protease/sample mixture is non-hypotonic;   c) filtering the mixture obtained in step (b) through a filter suitable for retaining microbial cells; and   d) recovering the microbial cells retained by the filter in step (c), wherein the recovered microbial cells are viable.   
     
     
         2 . The method of  claim 1 , wherein the sample is contacted with the buffer solution and protease(s) separately. 
     
     
         3 . The method of  claim 1 , wherein the buffer solution and protease(s) are mixed prior to step (b). 
     
     
         4 . The method of  claim 1 , wherein the sample is or comprises a clinical sample. 
     
     
         5 . The method of  claim 1 , wherein the sample is a clinical, biological, or environmental sample added to a medium. 
     
     
         6 . The method of  claim 5 , wherein the medium is a culture medium. 
     
     
         7 . The method of  claim 4 , wherein the clinical sample is a blood sample. 
     
     
         8 . The method of  claim 7 , wherein said blood sample is collected in a blood culture flask. 
     
     
         9 . The method of  claim 1 , wherein said protease is proteinase K. 
     
     
         10 . The method of  claim 1 , wherein the pH of the buffer solution is between pH 7 and less than pH 10.7. 
     
     
         11 . The method of  claim 1 , wherein the pH of the buffer solution is between pH 7 and pH 10.5. 
     
     
         12 . The method of  claim 1 , wherein the pH of the buffer solution is between pH 9 and pH 10.5. 
     
     
         13 . The method of  claim 1 , wherein the filter is a membrane filter. 
     
     
         14 . The method of  claim 1 , wherein the filter is a polyamide filter. 
     
     
         15 . The method of  claim 1 , wherein the filter has a pore size of less than 0.4 μm, preferably less than 0.22 μm. 
     
     
         16 . The method of  claim 1 , wherein the cells retained on the filter are recovered through back-flushing the filter using a liquid 
     
     
         17 . The method of  claim 16 , wherein the cells are back-flushed using culture medium. 
     
     
         18 . The method of  claim 1 , wherein said method further comprises one or more wash steps between steps (c) and (d). 
     
     
         19 . The method of  claim 18 , wherein said wash step selectively lyses non-microbial cells retained on the filter. 
     
     
         20 . A microbial recovery device for the recovery of viable microbial cells from a complex sample, said device being arranged to receive a complex sample of at least 1 ml, the device comprising: a reservoir comprising a buffer solution containing one or more proteases, or a reservoir containing a buffer solution and a reservoir containing one or more proteases, wherein said buffer solution has a pH of at least pH 6 and less than pH 11, wherein said buffer and said proteases do not comprise a detergent or a chaotrope, and wherein the microbial recovery device is arranged for mixing the sample with the buffer solution and proteases to produce a mixture; a filter suitable for retaining microbial cells and arranged to receive and filter the mixture; and a reservoir containing a liquid for recovering viable microbial cells from the filter, the reservoir connected to the filter and the microbial recovery device being arranged to convey the liquid to the filter to remove the retained microbial cells. 
     
     
         21 . The device of  claim 20 , wherein the reservoir containing a liquid for recovering viable microbial cells is connected to the opposite side of the filter to the sample chamber. 
     
     
         22 . The device of  claim 20 , wherein the device further comprises a reservoir containing a wash buffer for washing the filter after filtration, wherein the reservoir is connected to the filter. 
     
     
         23 . The device of  claim 20 , wherein the device is configured to perform the method of  claim 1 .