# US 7,608,419 B2 · Method and apparatus for detecting and quantifying bacterial spores on a surface > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,608,419 B2 - Document ID: US7608419B2 - Status: Granted - Kind: B2 - Application number: 10/987,202 - Filed: 2004-11-12 - Published: 2005-06-23 - Granted: 2009-10-27 - Earliest claimed: 2003-11-13 - Assignees: California Institute of Technology - Inventors: Adrian Ponce - Web page: https://patentgrep.com/patent/US7608419B2 ## CPC - C12Q 1/22: Testing for sterility conditions - C12Q 1/06: Quantitative determination - G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks} - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract A method and an apparatus for detecting and quantifying bacterial spores on a surface. In accordance with the method: a matrix including lanthanide ions is provided on the surface containing the bacterial spores; functionalized aromatic molecules are released from the bacterial spores on the surface; a complex of the lanthanide ion and the aromatic molecule is formed on the surface; the complex of the lanthanide ion and the aromatic molecule is excited to generate a characteristic luminescence of the complex on the surface; and the bacterial spores exhibiting the luminescence of the complex on the surface are detected and quantified. ## Claims ### Claim 1 A method for detecting and quantifying individual bacterial spores on a test surface comprising: providing a matrix including one or more lanthanide ions on the test surface containing the bacterial spores; releasing functionalized aromatic molecules from the bacterial spores onto the surface; forming a complex of the lanthanide ion and the aromatic molecule on the test surface; exciting the complex of the lanthanide ion and the aromatic molecule to generate a characteristic luminescence of the complex on the test surface; and detecting and quantifying, through lifetime-gated imaging, the individual bacterial spores exhibiting the luminescence of the complex on the test surface. ### Claim 2 (depends on 1) The method according to claim 1, wherein one or more lanthanide ions are provided to the matrix prior to the bacterial spores, after the bacterial spores. ### Claim 3 (depends on 1) The method according to claim 1, wherein the provided lanthanide ions are terbium or europium ions or a mixture thereof. ### Claim 4 (depends on 1) The method according to claim 1, wherein the released aromatic molecules are dipicolinic acid (DPA) and/or derivatives thereof. ### Claim 5 (depends on 1) The method according to claim 1, wherein the aromatic molecules are released from the bacterial spores by microwaving the bacterial spores. ### Claim 6 (depends on 1) The method according to claim 1, wherein the aromatic molecules are released from the bacterial spores by germinating the bacterial spores with L-alanine, inosine and mixtures thereof. ### Claim 7 (depends on 1) The method according to claim 1, wherein the aromatic molecules are released from the bacterial spores by sonicating the bacterial spores with microspheres. ### Claim 8 (depends on 1) The method according to claim 1, wherein the aromatic molecules are released from the bacterial spores by autoclaving. ### Claim 9 (depends on 1) The method according to claim 1, wherein the complex of the lanthanide ion and the aromatic molecule is excited by UV light. ### Claim 10 (depends on 1) The method according to claim 1, wherein the lifetime-gated imaging comprises counting bright spots due to DPA triggered luminescence around the bacterial spores shown in images obtained by a camera, preferably mounted on top of a microscope. ### Claim 11 (depends on 10) The method according to claim 10, wherein the imaging shows the specific resolution of the concentration of dipicolinic acid around the individual bacterial spores, which release dipicolinic acid. ### Claim 12 A method for detecting and quantifying individual bacterial spores on a surface comprising: transferring the bacterial spores from the surface containing bacterial spores to a test surface; providing a matrix including lanthanide ions on the test surface; releasing functionalized aromatic molecules from the bacterial spores on the test surface; forming complexes of the lanthanide ions and the aromatic molecules on the test surface; exciting the complexes of the lanthanide ions and the aromatic molecules to generate a characteristic luminescence of the complexes on the test surface; and detecting and quantifying through lifetime-gated imaging, the individual bacterial spores exhibiting the luminescence of the complexes on the test surface. ### Claim 13 (depends on 12) The method according to claim 12, wherein the test surface contains at least one polymer. ### Claim 14 (depends on 12) The method according to claim 12, wherein the test surface contains at least one adhesive polymer. ### Claim 15 (depends on 12) The method according to claim 12, wherein the test surface contains at least one adhesive polymer, which is transparent until about 250 nm, thereby providing a transparent test surface. ### Claim 16 (depends on 12) The method according to claim 12, wherein the test surface contains at least one lanthanide ions and L-alanine doped polymer. ### Claim 17 (depends on 12) The method according to claim 12, wherein the test surface contains at least one lanthanide ion and L-alanine doped polydimethylsiloxane polymer. ### Claim 18 (depends on 12) The method according to claim 12, wherein the test surface contains at least one agar. ### Claim 19 (depends on 12) The method according to claim 12, wherein the provided lanthanide ions are terbium or europium ions or a mixture thereof. ### Claim 20 (depends on 12) The method according to claim 12, wherein the released aromatic molecules are dipicolinic acid (DPA). ### Claim 21 (depends on 12) The method according to claim 12, wherein the aromatic molecules are released from the bacterial spores by microwaving the bacterial spores. ### Claim 22 (depends on 12) The method according to claim 12, wherein the aromatic molecules are released from the bacterial spores by germinating the bacterial spores with L-alanine. ### Claim 23 (depends on 12) The method according to claim 12, wherein the aromatic molecule are released from the bacterial spores by sonicating the bacterial spores with microspheres. ### Claim 24 (depends on 12) The method according to claim 12, wherein the complex of the lanthanide ion and the aromatic molecule is excited by UV light. ### Claim 25 (depends on 12) The method according to claim 12, wherein the imaging shows the specific resolution of the concentration of dipicolinic acid around bacterial spores, which release dipicolinic acid. ### Claim 26 (depends on 1) The method according to claim 1, wherein the matrix containing bacterial spores comprises at least one air filter. ### Claim 27 (depends on 12) The method according to claim 12, wherein the matrix containing bacterial spores comprises at least one air filter. ### Claim 28 (depends on 1) The method according to claim 1, wherein the matrix containing bacterial spores comprises at least one water filter. ### Claim 29 (depends on 12) The method according to claim 12, wherein the matrix containing bacterial spores comprises at least one water filter. ### Claim 30 An apparatus for detecting and quantifying individual bacterial spores on a surface including lanthanide ions and aromatic molecules released from the bacterial spores on the surface comprising: a test surface having thereon bacterial spores, lanthanide ions and aromatic molecules released from the bacterial spores, wherein the aromatic molecules have been released on the test surface by germination of bacterial spores by the addition of L-alanine, inosine or mixtures thereof or the lysis of bacterial spores by means of one selected from autoclaving, microwaving, heating and sonication; a UV-light radiation device for exciting a complex of lanthanide ion and aromatic molecule to generate a characteristic luminescence of the complex on the test surface; a microscope for detecting and quantifying bacterial spores exhibiting the luminescence of the complex on the test surface; and a lifetime-gated imaging device for imaging bacterial spores exhibiting the luminescence. ### Claim 31 (depends on 1) The method according to claim 1, wherein one or more lanthanide ions are provided as a mixture with the bacterial spores.