Method and apparatus for detecting and quantifying bacterial spores on a surface

Abstract

A method and an apparatus for detecting and quantifying bacterial spores on a surface. In accordance with the method: bacterial spores are transferred from a place of origin to a test surface, the test surface comprises lanthanide ions. Aromatic molecules are released from the bacterial spores; a complex of the lanthanide ions and aromatic molecules is formed on the test surface, the complex is excited to generate a characteristic luminescence on the test surface; the luminescence on the test surface is detected and quantified.

1 .- 31 . (canceled) 
     
     
         32 . A method for quantifying viable and non-viable bacterial spores in a population of individual bacterial spores, the method comprising:
 applying one or more lanthanide ions and the population of individual bacterial spores on a test surface;   releasing first aromatic molecules from first individual bacterial spores in the population of individual bacterial spores by germination of the first individual bacterial spores on the test surface;   forming a first complex of the one or more lanthanide ions and the first aromatic molecules on the test surface;   exciting the first complex to generate a characteristic luminescence of the first complex on the test surface;   detecting and quantifying the first individual bacterial spores exhibiting the luminescence of the first complex on the surface;   releasing second aromatic molecules from second non-germinated individual bacterial spores in the population of individual bacterial spores on the test surface by lysis;   forming a second complex of the one or more lanthanide ions and the second aromatic molecules on the test surface;   exciting the second complex to generate a characteristic luminescence of the second complex on the test surface; and   detecting and quantifying the second non-germinated individual bacterial spores represented by the luminescence of the second complex on the test surface by lifetime-gated imaging.   
     
     
         33 . The method according to  claim 32 , wherein the applying the population of individual bacterial spores on the test surface comprises first capturing the population of individual bacterial spores, and then transferring the population of individual bacterial spores to the test surface. 
     
     
         34 . The method according to  claim 33 , wherein the capturing the population of individual bacterial spores comprises collecting the population of individual bacterial spores in an adhesive polymer. 
     
     
         35 . The method of  claim 34 , wherein the adhesive polymer is selected from the group consisting of PDMS, agar and agarose. 
     
     
         36 . The method of  claim 33 , wherein the capturing of the population of individual bacterial spores comprises collecting the population of individual bacterial spores with a swab. 
     
     
         37 . The method of  claim 36 , further comprising transferring the population of individual bacterial spores from the swab into water. 
     
     
         38 . The method of  claim 37 , further comprising passing the water with the population of individual bacterial spores through a water filter. 
     
     
         39 . The method of  claim 33 , wherein the capturing of the population of individual bacterial spores comprises collecting the population of individual bacterial spores from water and/or air. 
     
     
         40 . The method of  claim 39 , further comprising transferring the population of individual bacterial spores from the water and/or air to an air filter and/or a water filter. 
     
     
         41 . The method of  claim 32 , wherein the test surface comprises PDMS, agar, agarose, PDMS together with agar, PDMS together with agarose, or a combination thereof. 
     
     
         42 . The method of  claim 32 , wherein the test surface comprises at least one partially transparent adhesive polymer. 
     
     
         43 . The method of  claim 32 , wherein the one or more lanthanide ions are provided to the test surface prior to the population of individual bacterial spores, after the population of individual bacterial spores, or as a mixture with the population of individual bacterial spores. 
     
     
         44 . The method of  claim 32 , wherein the one or more lanthanide ions comprises terbium and/or europium. 
     
     
         45 . The method of  claim 32 , wherein the first and/or second aromatic molecules comprise dipicolinic acid, dipicolinate, dipicolinic acid together with dipicolinate, or a combination thereof. 
     
     
         46 . The method of  claim 32 , wherein the first and/or second aromatic molecules interact with the test surface resulting in one or more derivatives of the first and/or second aromatic molecules. 
     
     
         47 . The method of  claim 32 , wherein the germination of the first individual bacterial spores is induced by a germinating agent selected from the group consisting of L-alanine, L-asparagine, 0-glucose, and combinations thereof. 
     
     
         48 . The method of  claim 32 , wherein the lysis of the second non-germinated individual bacterial spores is induced by microwaving, autoclaving, sonication, plasma cleaning dry heating, and/or hydrogen chloride gassing. 
     
     
         49 . The method according to  claim 32 , wherein the exciting the first complex and/or the exciting the second complex comprises excitation by UV light. 
     
     
         50 . The method according to  claim 32 , further comprising quantifying the percent viable spores in the population of bacterial spores by:
 detecting and quantifying the first individual bacterial spores represented by the luminescence of the first complex; and   dividing the quantified first individual bacterial spores by the total number of bacterial spores in the population of individual bacterial spores.   
     
     
         51 . The method according to  claim 32 , further comprising quantifying the percent viable bacterial spores in the population of bacterial spores by:
 detecting and quantifying the first individual bacterial spores represented by the luminescence of the first complex;   detecting and quantifying the second non-germinated individual bacterial spores represented by the luminescence of the second complex; and   dividing the quantified luminescence of the first complex by the sum of the quantified luminescence of the first and second complexes.   
     
     
         52 . A method of  claim 51 , wherein the quantifying the first individual bacterial spores and/or the second non-germinated individual bacterial spores represented by the respective luminescence comprises counting the number of luminescent spots and estimating the number of individual bacterial spores per luminescent spot based on the spot intensity.