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: 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

1. A method for detecting and quantifying individual bacterial spores, the method comprising imaging the individual bacterial spores on a matrix comprising a lanthanide ion, wherein the imaging is performed following release of aromatic molecules from the individual bacterial spores and subsequent excitement of a complex formed by the lanthanide ion and the aromatic molecules to generate a luminescence; and wherein the imaged individual bacterial spores exhibit the luminescence of the complex on the matrix.

2. The method according to claim 1, wherein the matrix further comprises at least one polymer.

3. The method according to claim 2, wherein the at least one polymer is an adhesive polymer.

4. The method according to claim 3, wherein the at least one adhesive polymer is transparent down to about 250 nm, thereby providing a transparent test surface.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A is a microscopic image of a spore (about 1 μm in diameter) highlighting a DPA rich spore core.

(2) FIG. 1B is a diagram of a Tb.sup.3+ ion (shaded ball) which by itself has a low absorption cross section (<10 M.sup.−1 cm.sup.−1) and consequently has low luminescence intensity. The Tb.sup.3+ ion can bind the light harvesting DPA (absorption cross section >10.sup.4 M.sup.−1) originating from the spore. DPA binding gives rise to bright Tb luminescence.

(3) FIG. 1C is a diagram of a photophysical scheme for DPA sensitized luminescence of the Tb complex (absorption-energy transfer-emission, AETE).

(4) FIG. 2 depicts a photograph of a backlight illuminated quartz slide with three solidified agar drops. (A) No Tb.sup.3+ added. (B) Tb.sup.3+ added but no L-alanine (C)Tb.sup.3++L-alanine after germination completion.

(5) FIG. 3 depicts Eu.sup.3+ microspheres (1-μm) on fluorescent paper imaged with an Imagex-TGi gated CCD camera mounted on a Cart Zeiss fluorescence microscope with 40× objective, excited with a 300-Hz Perkin Elmer flashlamp. Images are obtained (A) without gating, (B) with gating (100-μs delay, 2.7-ms gate), and (C) 100-μm reference graticule to estimate spatial resolution.

(6) FIG. 4 depicts a schematic apparatus for imaging quantifying and counting of bacterial spores.

(7) FIG. 5 depicts two lifetime gated photographs showing bacterial spores on R2A agar before germination (left portion of the figure) and after germination (right portion of the figure).

DETAILED DESCRIPTION

(8) Bacterial spores are generally accepted to be indicator species for validating sterility since they are the most resilient form of life towards sterilization regimens. Sterility testing of surfaces is traditionally performed with RODAC growth plates that require 3-5 days before results are available. The method and apparatus according to the present disclosure will yield results within minutes for obtaining total bacterial spore counts, and an hour for obtaining viable bacterial spore counts on surfaces.

(9) Dipicolinic acid (DPA, 2,6 pyridinedicarboxylic acid) is present in high concentrations (about 1 molar or about 15% of by weight) in the core of bacterial spores 38 as a 1:1 complex with Ca.sup.2+ as shown in FIG. 1a. For all known lifeforms, DPA is unique to bacterial spores and is released into bulk solution upon germination, which is the process of spore-to-vegetative cell transformation. Thus, DPA is an indicator molecule for the presence of bacterial spores. DPA is also a classic inorganic chemistry ligand that binds metal ions with high affinity. DPA binding to terbium ions (or other luminescent lanthanide or transition metal ions) triggers intense green luminescence under UV excitation as shown in FIGS. 1b and 1c. The green luminescence turn-on signal indicates the presence of bacterial spores. The intensity of the luminescence can be correlated to the number of bacterial spores per milliliter.

(10) The Tb-DPA luminescence assay can be employed to detect bacterial spores on surfaces, including the surfaces of air filters, water membrane filters, and adhesive polymers or agar used to collect bacterial spores from surfaces to be tested. In this disclosure, surfaces to be analyzed with the Tb-DPA assay are called “test surfaces”. For example, the Tb-DPA luminescence assay can be combined with an optically transparent, adhesive polymer or agar to collect bacterial spores from surfaces to be tested. Once the bacterial spores are located on the test surface, they can be induced to release their DPA content by germination or physical lysis, for example by autoclaving or microwaving. The highly concentrated DPA from the spores spills into the surrounding area, generating a high concentration region around the spore body. The reagents used for detection and induction of germination, if that is the chosen method for DPA release, can be added into the matrix before or after the spores are sampled. The Tb-DPA luminescence arising from the region around the spore body is then imaged onto a camera. The bacterial spore regions manifest themselves as bright spots which can be counted. Due to the long-lived excited states of luminescent lanthanides, lifetime-gated detection enables any fluorescent background from interferrents to be elimated. Lifetime gating drastically reduces the background and enables much greater contrast between the Tb-DPA luminescence regions and the background.

(11) One example of an adhesive polymer for the Tb-DPA luminescence assay for bacterial spores on surfaces is polydimethyl siloxane (PDMS) doped with TbCl.sub.3 and L-alanine. The L-alanine induces germination to release the DPA from the core of the spore to the immediate surroundings. The TbCl.sub.3 binds the DPA, which triggers green luminescence (543.5 nm) under UV excitation (250-300 nm) that can be quantified with a photodetector. Specifically, imaging individual germinating spores within a microscope field of view using a lifetime-gated camera will be used as an example.

(12) One example of an adhesive polymer for the Tb-DPA luminescence assay for bacterial spores on surfaces is polydimethyl siloxane (PDMS) doped with TbCl.sub.3 and L-alanine. The L-alanine induces germination to release the DPA from the core of the spore to the immediate surroundings. The TbCl.sub.3 binds the DPA, which triggers green luminescence (543.5 nm) under UV excitation (250-300 nm) that can be quantified with a photodetector. Specifically, we will use the example of imaging individual germinating spores within a microscope field of view using a lifetime-gated camera.

(13) From the perspective of our sensor design, we treat the bacterial spore essentially as a ˜1-μm sphere containing ˜10.sup.9 molecules of DPA. In our previous experiments, we collected spores from surfaces using the standard cotton swabbing method, resuspended the spores into water, and then released the DPA contents into bulk solution by germination or physical lysing and subsequently performed the Tb-DPA luminescence assay. This approach led to very dilute DPA solutions (e.g., 1 spore per ml of solution yields [DPA]=1 pM), which ultimately limits the sensitivity.

(14) Instead of diluting the DPA into bulk solution, we immobilize the bacterial spores onto an adhesive polymer (e.g., PDMS), and then induce germination or physically lysis in the spore population on the polymer to generate local high DPA concentrations (i.e, the DPA remains in the immediate surroundings of the spore body). To obtain viable counts, germination will be induced by doping L-alanine (or other germination inducing agents) into the polymer matrix; TbCl.sub.3, also doped into the polymer, report the presence of bacterial spores by triggering luminescence in the presence of DPA. To obtain total counts, the bacterial spores immobilized on the TbCl.sub.3 containing polymer will be physical lysed (e.g., by heat, microwaving, or autoclaving) leads to DPA release and luminescence turn-on.

(15) The present disclosure also includes a method and apparatus to measure the fraction of bacterial spores that remain viable or alive, hence a live/dead assay for bacterial spores. The method combines dipicolinic acid triggered terbium luminescence and dipicolinic acid release from (1) viable bacterial spore through germination, and (2) all viable and nonviable bacterial spores by autoclaving, sonication, or microwaving. The ratio of the results from steps (1) and (2) yield the fraction of bacterial spores that are alive.

(16) The traditional culture based assays require 3 days for colonies to grow and be counted. However, a significant fraction of bacterial spores can undergo stage-1 germination, during which DPA (i.e., the chemical marker that is unique to bacterial spores) is released, in less than 40 minutes. See FIG. 2. A DPA-triggered Tb luminescence with Tb-doped agar was investigated. The samples were prepared by adding ˜100 μl of agar doped with 1 mM TbCl.sub.3 onto a quartz slide and allowing it to solidify. On top of the agar, we added 10 μl of 10.sup.9 spores/ml Bacillus subtilis spores (i.e., 10.sup.7 spores), and then added a drop of 10 μl of 1-mM L-alanine to induce germination.

(17) Under UV (blacklight) illumination, the luminescence of the embedded Tb increased dramatically upon germination within 40 minutes of the bacterial spores, while the embedded Tb luminescence in the control sample that had no exposure to L-alanine remained weak. See FIG. 2. An agar control sample without Tb that was covered with bacterial spores also did not yield detectable luminescence. Note that the bright edges of the spots are artifacts of drying due to refraction from accumulated material, which would not appear in a lifetime-gated image.

(18) The pictures in FIG. 2 were taken without magnification, and thus the individual spores cannot be enumerated as they germinate. However, in the proposed effort, germinating bacterial spores will be imaged with a lifetime-gated microscope. As the spores germinate, DPA is released from the core to generate local high DPA concentrations, which will show up as bright green luminescent halos surrounding the spore body. These results demonstrate that viable bacterial spores on surfaces by employing the JPL Endospore Viability Assay can be enumerated.

(19) Lifetime-gated images of Eu.sup.3+ microspheres on highly fluorescent paper were obtained with a lifetime-gated camera (Photonic Research Systems Ltd, United Kingdom). See FIG. 3. Eu.sup.3+ microspheres were employed because they are commercially available and have analogous photophysical properties. The Imagex system effectively rejected all of the strong background fluorescence when a delay time of 100 μs was used. It is striking that the microspheres exhibiting weak, long-lived luminescence immobilized on a highly fluorescent matrix are imaged with high contrast against a silent background when gating is applied.

(20) Another example of the invention is illustrated in FIG. 5, where bacterial spores were added onto the surface of R2A agar doped with 10 mM L-alanine to induce germination and 100 uM TbCl.sub.3 to generate bright luminescent spots around the spore body as they germinated and released DPA. A Xe-flash lamp firing at 300 Hz with a 275 nm interference filter provided excitation for the Tb-DPA complex, and the corresponding bright spots from the bacterial spore Tb-DPA luminescent halos where imaged with a lifetime-gated camera set at a delay time of 100 μs and an integration time of 2 ms. The individual bacterial spores become clearly visible as countable spots after they germinated. The images shown in FIG. 5 can be obtained by an apparatus as shown in FIG. 4, which contains a Xenon flash lamp, a microscope objective, a microscope, and a lifetime gated camera mounted on the microscope.

EXAMPLES

Comparative Example 1 Performed According to U.S. Pub. App. No. 2004-0014154

(21) Aerosolized bacterial spores were captured with an aerosol biosampler. The biosampler was filled with 20 ml of 10 μM TbCl.sub.3 glycerol solution, which has a 95% transfer efficiency for microbe-containing aerosols. Once bacterial spores were suspended in the biosampler collection vessel, DPA was released by microwave into the bulk solution within 8 minutes. The resulting free DPA then bounded Tb in bulk solution, giving rise to luminescence turn-on under UV excitation. A fiber optic probe immersed in the sample solution transmitted the luminescence to a spectrometer.

(22) Approximately 10,000 bacterial spores per 1 ml solution produced enough DPA to obtain sufficient amount of DPA-Tb complexes to provide enough luminescence turned-on under UV excitation to be detected by a spectrometer.

Comparative Example 2 Performed According to U.S. Pub. App. No. 2004-0014154

(23) Comparative Example 2 was performed like Comparative Example 1. A fiber optic probe immersed in the sample solution transmitted the luminescence to a fluorimeter.

(24) Approximately 1,000,000 bacterial spores per 1 ml solution produced enough DPA to obtain sufficient amount of DPA-Tb complexes to provide enough luminescence turned-on under UV excitation to be detected by a spectrometer.

Example 1

(25) Bacteria spores were immobilized onto a test sample surface of thin, flexible, clear, adhesive polymer polydimethylsiloxan (PDMS). PDMS was doped with L-alanine to induce germination and generate local high concentration of DPA. TbCl.sub.3 was also doped into the PDMS sample. The bacterial spores immobilized on the L-alanine and TbCl.sub.3 containing polymer was physically lysed by microwave irradiation, wherein DPA was released and luminescence was turned on. The detection of bacterial spores on the PDMS adhesive polymer was manifested itself as a bright green luminescence that was imaged with a lifetime gated microscope. The green dots within the microscope field of view were counted to determine the concentration of viable spores found on the surfaces that was sampled. Therefore, every bacterial spore releasing luminescence can be individually counted. A concentration of 10,000 bacterial spores per 1 ml as in comparative example 1 or 1,000,000 bacterial spores per 1 ml in comparative example 2 is not required in example 1. As a consequence, the method according to the disclosure can be carried out even with an extremely low concentration of bacterial spores, even a single bacterial spore.

(26) Another embodiment of the present invention is an apparatus for detecting and quantifying bacterial spores on a surface including lanthanide ions and aromatic molecules released from the bacterial spores on the surface. See FIG. 4. The apparatus comprises an UV-light radiation device for exciting a complex of a lanthanide ion and an aromatic molecule to generate a characteristic luminescence of the complex on a surface. The source for the UV-light is preferably a Xenon flash lamp, which is approximately 5 cm away the test surface. Between the Xenon flash lamp and the test surface are two C-amount elliptical lenses. The Xenon flash lamp and the test substrate are positioned in an angel of 45° to each other. The area of irradiation by the Xenon flash lamp is observed by a microscope objective with a red bandpass filter suitable for Eu.sup.3+ for detecting and quantifying bacterial spores exhibiting the luminescence of the complex on the surface. The image is transferred from the microscope to the imaging devise for imaging bacterial spores exhibiting the luminescence, preferably an imageX nanoCCD camera.

(27) The method and apparatus of the present disclosure provide the imaging of the spherical resolution of the high concentrating region of DPA around each spore body, which has been lysed. The present method makes it possible to detect and quantify extremely low concentrations of bacterial spores in very short time.

(28) Bioburden testing is an assessment of the numbers and types of microorganisms present on a product, and may be used to support sterilization validations. Sterility determination for surfaces are required by the pharmaceutical, health care, and food preparation industries for compliance with bioburden standards as outlined by USP, FDA, PDA, and AAMI.

(29) While several illustrative embodiments have been shown and described in the above description, numerous variations and alternative embodiments will occur to those skilled in the art. Such variations and alternative embodiments are contemplated, and can be made without departing from the scope of the invention as defined in the appended claims.