Apparatus and method for pretreatment of microbial samples

Abstract

Methods and devices are provided for pretreatment of a sample containing microbial cells. In some embodiments, the pretreatment of the sample is performed via the initial selective lysis, within a sample pretreatment vessel, of non-microbial cells (such as blood cells) and the subsequent centrifugal separation of the sample to remove the resulting debris and concentrate the microbial cells. An immiscible and dense cushioning liquid may be included for collecting the microbial cells adjacent to the liquid interface formed by the cushioning liquid upon centrifugation of the pretreatment vessel. After removal of a substantial quantity of the supernatant, resuspension of the collected microbial cells, and re-establishment of the cushioning liquid interface, at least a portion of the remaining suspension may be removed without substantially removing the cushioning liquid. One or more intermediate wash cycles may be performed prior to extraction of the remaining suspension, which provides a “pretreated” sample.

1 - 4 . (canceled) 
     
     
         5 . An automated system for processing a positive blood culture sample, the automated system comprising:
 a centrifugal processing receptacle comprising:
 a main body having a proximal opening and a closed distal end, said main body comprising an inner chamber accessible though the proximal opening, wherein a surface defining said inner chamber comprises a first surface and a second surface, wherein said second surface is conical in shape and extends distalward relative to said first surface, and wherein a proximal portion of said second surface resides adjacent to a distal portion of said first surface; and 
 a cap for closing the proximal opening during centrifugation of said centrifugal processing receptacle; 
   a rotor assembly configured to support said centrifugal processing receptacle;   a liquid handling subsystem capable of automated dispensing and aspiration;   control hardware operatively coupled to said rotor assembly and said liquid handling subsystem, said control hardware being configured to perform operations comprising:
 controlling said liquid handling subsystem to combine the positive blood culture sample with a lysis reagent to form a mixture within said inner chamber of said centrifugal processing receptacle; 
 controlling said rotor assembly to centrifuge said centrifugal processing receptacle with the mixture residing within said inner chamber and the proximal opening closed by said cap, such that microbial cells are collected within a collection region residing between the proximal portion of said second surface and the distal portion of said first surface; 
 controlling said liquid handling subsystem to access said inner chamber and aspirate a first supernatant from said inner chamber; 
 controlling said liquid handling subsystem to dispense a wash buffer into said inner chamber; 
 controlling said rotor assembly to centrifuge said centrifugal processing receptacle with the wash buffer and the microbial cells residing within said inner chamber of said centrifugal processing receptacle and the proximal opening closed by said cap; and 
 controlling said liquid handling subsystem to access said inner chamber and aspirate a second supernatant from said inner chamber, such that said inner chamber comprises purified and concentrated microbial cells. 
   
     
     
         6 . The automated system according to  claim 5  wherein said control hardware is further configured to control said rotor assembly to resuspend the microbial cells in the wash buffer after dispensing the wash buffer into said inner chamber. 
     
     
         7 . A method of processing a positive blood culture sample, the method comprising:
 providing a centrifugal processing receptacle comprising:
 a main body having a proximal opening and a closed distal end, the main body comprising an inner chamber accessible though the proximal opening, wherein a surface defining the inner chamber comprises a first surface and a second surface, wherein the second surface is conical in shape and extends distalward relative to the first surface, and wherein a proximal portion of the second surface resides adjacent to a distal portion of the first surface; and 
 a cap for closing the proximal opening during centrifugation of the centrifugal processing receptacle; 
   employing a liquid handling subsystem to form a mixture comprising the positive blood culture sample and a blood lysis reagent within the inner chamber;   employing a rotor assembly configured to support the centrifugal processing receptacle to centrifuge the centrifugal processing receptacle with the proximal opening closed by the cap, such that microbial cells are collected within a collection region residing between the proximal portion of the second surface and the distal portion of the first surface;   employing the liquid handling subsystem to access the inner chamber and aspirate a first supernatant from the inner chamber;   employing the liquid handling subsystem to dispense a wash buffer into the inner chamber;   employing the rotor assembly to centrifuge the centrifugal processing receptacle with the wash buffer and the microbial cells residing within the inner chamber of the centrifugal processing receptacle and the proximal opening closed by the cap; and   employing the liquid handling subsystem to access the inner chamber and aspirate a second supernatant from the inner chamber, such that the inner chamber comprises purified and concentrated microbial cells.   
     
     
         8 . The method according to  claim 7  further comprising resuspending the microbial cells in the wash buffer after dispensing the wash buffer into the inner chamber. 
     
     
         9 . An automated system for processing a liquid containing microbial cells, said automated system comprising:
 a centrifugal processing receptacle comprising:
 a main body having a proximal opening and a closed distal end, said main body comprising an inner chamber accessible though the proximal opening, wherein a surface defining said inner chamber comprises a first surface and a second surface, wherein said second surface is conical in shape and extends distalward relative to said first surface, and wherein a proximal portion of said second surface resides adjacent to a distal portion of said first surface; and 
 a cap for closing the proximal opening during centrifugation of said centrifugal processing receptacle; and 
   a rotor assembly configured to support said centrifugal processing receptacle; and   control hardware operatively coupled to said rotor assembly, said control hardware being configured to perform operations comprising:
 controlling said rotor assembly to centrifuge said centrifugal processing receptacle with the liquid residing within said inner chamber and the proximal opening closed by said cap, such that the microbial cells are collected within a collection region residing between the proximal portion of said second surface and the distal portion of said first surface. 
   
     
     
         10 . The automated system according to  claim 9  further comprising:
 a liquid handling subsystem capable of automated dispensing and aspiration, said control hardware being operatively coupled to said liquid handling subsystem; 
 said control hardware being operatively coupled to said liquid handling subsystem and further configured to perform operations comprising:
 controlling said liquid handling subsystem to access said inner chamber and aspirate a first supernatant from said inner chamber; 
 controlling said liquid handling subsystem to dispense a wash buffer into said inner chamber; 
 controlling said rotor assembly to centrifuge said centrifugal processing receptacle with the wash buffer and the microbial cells residing within said inner chamber of said centrifugal processing receptacle and the proximal opening closed by said cap; and 
 controlling said liquid handling subsystem to access said inner chamber and aspirate a second supernatant from said inner chamber, such that said inner chamber comprises purified and concentrated microbial cells. 
 
 
     
     
         11 . The automated system according to  claim 10  wherein said control hardware is further configured to control said rotor assembly to resuspend the microbial cells in the wash buffer after dispensing the wash buffer into said inner chamber. 
     
     
         12 . The automated system according to  claim 10  wherein said control hardware is configured to control said liquid handling subsystem to provide the liquid within said inner chamber by mixing a blood sample with a lysis reagent configured to facilitate lysis of blood cells within the blood sample. 
     
     
         13 . The automated system according to  claim 12  wherein the lysis reagent is configured for use with a positive blood culture sample. 
     
     
         14 . The automated system according to  claim 12  wherein the lysis reagent is configured for use with a whole blood sample. 
     
     
         15 . A method of processing a blood sample containing microbial cells, the method comprising:
 providing a centrifugal processing receptacle comprising:
 a main body having a proximal opening and a closed distal end, the main body comprising an inner chamber accessible though the proximal opening, wherein a surface defining the inner chamber comprises a first surface and a second surface, wherein the second surface is conical in shape and extends distalward relative to the first surface, and wherein a proximal portion of the second surface resides adjacent to a distal portion of the first surface; and 
 a cap for closing the proximal opening during centrifugation of the centrifugal processing receptacle; 
   forming a mixture comprising the blood sample and a blood lysis reagent within the inner chamber; and   employing a rotor assembly configured to support the centrifugal processing receptacle to centrifuge the centrifugal processing receptacle with the proximal opening closed by the cap, such that the microbial cells are collected within a collection region residing between the proximal portion of the second surface and the distal portion of the first surface.   
     
     
         16 . The method according to  claim 15  further comprising:
 employing a liquid handling subsystem to access the inner chamber and aspirate a first supernatant from the inner chamber; 
 employing the liquid handling subsystem to dispense a wash buffer into the inner chamber; 
 employing the rotor assembly to centrifuge the centrifugal processing receptacle with the wash buffer and the microbial cells residing within the inner chamber of the centrifugal processing receptacle and the proximal opening closed by the cap; and 
 employing the liquid handling subsystem to access the inner chamber and aspirate a second supernatant from the inner chamber, such that the inner chamber comprises purified and concentrated microbial cells. 
 
     
     
         17 . The method according to  claim 16  further comprising resuspending the microbial cells in the wash buffer after dispensing the wash buffer into the inner chamber. 
     
     
         18 . The method according to  claim 16  further comprising employing the liquid handling subsystem to form the mixture within the inner chamber. 
     
     
         19 . The method according to  claim 18  wherein the blood sample is a positive blood culture sample. 
     
     
         20 . The method according to  claim 18  wherein the blood sample is a whole blood sample. 
     
     
         21 . The method according to  claim 18  wherein the blood sample comprises an anticoagulant.