Patent Application
20190106723
The present invention relates to apparatus, systems and methods for live microscopy, and providing conditions that permit detection and evaluation of microorganisms under conditions that preserve metabolic activity. Relatedly, the apparatus, system and method is particularly relevant to the detection of viable culturable microorganisms in a sample that may contain viable non-culturable organisms, non-viable cells, and debris.
Detection of disease-causing microorganisms in food and water is important for public health. The key indicator of disease risk is the number of organisms that can reproduce. The ability to reproduce is typically measured by the ability to grow in suitable culturable medium. The gold standard for counting the number of culturable microorganisms is plate counting. This method, however, is slow, expensive, and can be affected by aggregation and other problems. Plate counting also relies on the use of suitable medium on which the organism can grow.
Direct detection and enumeration of microorganisms in a sample of food or water, etc., would be desirable. Microscopy techniques allow for detection of microorganisms in samples of various substances. However, enumeration of the number of bacteria is a very unreliable proxy for the number of culturable organisms. This is because the population of microorganisms is not entirely or even mostly made of culturable organisms, but also includes dead cells, and viable non-culturable cells (hereinafter referred to as “VNC”). VNC are viable, but typically are less metabolically active than culturable cells. VNC may be the majority of viable cells in a population, and the proportion of viable cells that are VNC varies according to numerous conditions.
The presence of dead cells, VNC, and debris poses problems for detection and enumeration of culturable organisms. Dye-staining allows for detection of bacteria and differentiation between live and dead cells, but not between culturable cells and VNC.
WO 2010004567 A1 describes a method and kit for direct detection and enumeration of culturable microbial cells from a sample that may also contain VNC, dead cells, and debris. The method relies on differences between culturable, VNC, and dead cells in the rate at which signal emitting agents associate and disassociate with the membrane. Metabolically active, culturable, cells typically take up certain dyes at a faster rate than VNC or dead cells. The use of specific time points thereby permits distinction between culturable cells and all other cells and debris.
The method of WO 2010004567 is relatively fast, taking less than an hour. However, even several minutes under unfavourable conditions may alter the metabolic state of microorganisms. The present inventors therefore sought to develop an apparatus, and related systems and methods, that preserve the bacteria in a metabolically active state to permit accurate detection and enumeration.
In one embodiment, a system for detecting microbial cells in a sample includes an apparatus configured to image at least one cell in the sample. The apparatus comprises a holder comprising an internal portion and an external portion, the holder being configured to secure a member between the portions; an imaging device disposed above the external portion and configured to permit examination of the at least one cell; a stage attachable to a stage platform, the stage platform being configured to connect to a motor, and a projecting member projecting from an upper surface of the stage and configured to receive and provide solution. The system may further include a cooling device. The cooling device may comprise a thermoelectric cooling element disposed between the stage and the stage platform. Alternatively, cooling may be achieved by at least one inlet tube and at least one outlet tube configured to circulate a cooling medium beneath the stage, or by cooling device such as fans.
In another embodiment, a method for detecting microbial cells in a sample comprises filtering the sample through a membrane so as to capture a total number of cells in the sample on the membrane; applying the solution to the upper surface of the projecting member; fixing the holder to the projecting member such that the membrane is secured atop the projecting member contacts the solution and is supported by the upper surface of the projecting member; preparing the total number of cells for imaging by staining the total number of cells prior to cleansing, acquiring at least one image of the total number of cells in accordance with a sampling protocol, and detecting microbial cells.
In yet another embodiment, an apparatus for determining a number of culturable microbial cells in a sample comprises an internal ring and an external ring. The rings are configured so as to stretch a member between the rings and to maintain a position of the member relative to the rings. The apparatus further comprises an imaging device disposed above the external ring and configured to permit imaging of at least one cell in the sample; a stage attachable to a stage platform, the stage platform being configured to connect to a motor, and a projecting member projecting from an upper surface of the stage and configured to receive and provide solution, wherein a plurality of dimples provided on the stage are configured to engage with a plurality of pins on the stage platform.
The features, aspects and advantages of the present invention will become apparent from the following description, appended claims, and the accompanying exemplary embodiments shown in the drawings, which are briefly described below.
The following detailed description is exemplary and explanatory only, and is not restrictive of the invention as claimed. In the following detailed description, reference is made to the accompanying drawings, in which similar symbols typically identify similar components, unless context dictates otherwise.
The drawings described above depict an apparatus configured to permit detection of culturable cells in a particular sample. Detection may further permit enumeration of the number of culturable cells. Techniques for determining the number of culturable cells in a sample containing, for example, culturable microbial cells, viable non-culturable microbial cells, and non-viable microbial cells are disclosed in U.S. patent application Ser. No. 13/003,128 to Glukhman, the entire contents of which are incorporated herein by reference for background information and the systems, kits, programs, processes and techniques disclosed therein.
Detection of the number of viable, culturable cells in a sample is challenging for various reasons. Notably, treatment protocols employed for imaging of the bacteria cells, and environmental conditions of the image acquisition process can cause stress to the imaged cells or even be lethal to them. In such circumstances, the cells can change their characteristics, for example, their metabolic behavior. Traditional imaging techniques suffer from difficulties in accurately capturing the characteristics of cells in their natural, intact state prior to preparation for imaging.
As a further complication, imaging generally requires sustaining a metabolic condition of the cells, as exhibited prior to testing, during the imaging process. Thus, the environment in which the cells are kept during image acquisition must be tailored so as to preserve a pre-test metabolic condition of the cells. The sampling process may require that the pre-test metabolic condition be maintained for half an hour or more than an hour, for example. Although imaging itself may take several minutes, other processes associated with sampling (e.g., preparation of the sample) take additional time. Accordingly, an appropriate environment must be maintained for the requisite time to ensure that the cells remain viable and in their pre-test metabolic condition during image acquisition. The embodiments described below allow for such pre-test metabolic conditions to be preserved.
First, an overview of one embodiment of an apparatus for determining a number of culturable microbial cells in a sample will be described.
The imaging device comprises components that allow for microscopic imaging of the sample. In addition to the objective lens 10 shown in
More particularly, the imaging device allow for images to be taken of cells in the sample at the microscopic level. To this end, the cells in the sample are bound to a membrane 22. The membrane 22 is a membrane or filter. The holder 20 is configured to securely hold the membrane 22 in a fixed position in which the cells bound to the membrane are imaged by the imaging device.
The holder 20, in some embodiments, comprises a plurality of rings, as will be described below. In other embodiments, the holder 20 is composed of rectilinear elements. In at least one embodiment, the holder 20 is a clamp. The disposition of the membrane 22 inside the holder 20 is discussed in more detail below in the context of
Referring again to
The stage 32 is configured to be coupled to the stage platform 30 in a manner that permits movement of the stage 32 along a first axis and along a second axis. For example, with reference to a coordinate system as shown in the lower left corner of
Furthermore, although the stage 32 shown in
Turning now to
Further, in some embodiments, the stage platform 30 is configured to couple to and decouple from the imaging device. In some embodiments, the stage platform is configured to couple to and decouple from a motor (described below). In some embodiments, the stage platform 30 is configured to detach to the imaging device so as to extend from a first position to be in a second position. The second position, for example, may be beyond an enclosure of the imaging device, thus permitting the stage platform 30 to receive the membrane 22 and afterwards return to the first position. In some embodiments, the stage platform 30 is permanently fixed to the imaging device.
In some embodiments, the upper surface 36 of the projecting member 34 is configured to receive a solution, some amount of which is imparted to the membrane 22 when the membrane 22 contacts the projecting member 34. In some embodiments, the projecting member 34 comprises a solid cup-shaped member into which a sponge impregnated with the solution is inserted. In some embodiments, the projecting member 34 is provided with a tunnel or groove into which the solution is applied. When the projecting member 34 contacts the membrane 22, a quantity of the solution spreads to the membrane 22. In some embodiments, application of the solution comprises manually applying solution to the upper surface 36 of the projecting member 34. In some embodiments, application of the solution comprises directing the solution from a reservoir onto the upper surface 36 of the projecting member 34.
In certain embodiments, a fluid, for example, the aforementioned solution, can be exchanged so as to ensure that a fresh supply of fluid is provided to the membrane 22. The exchange of fluid can be accomplished via at least one microfluidic tube or at least one non-microfluidic tube. In some embodiments, at least one tube is provided to permit fluid to be exchanged on the upper surface 36 of the projecting member 34. In some embodiments, an exchange of fluid is accomplished via a plurality of tubes. In some embodiments, a groove, tube, tunnel, or channel is provided on the projecting member 34 so as to permit fluid exchange. Such configurations allow, in some embodiments, to permit fluid to be exchanged without a manual application of fluid on the membrane 22 or on the projecting member 34. In some embodiments, fluid exchange allows for staining and/or washing of cells on the membrane 22.
Further, in such embodiments, when the projecting member 34 contacts the holder 20, an amount of solution contacts the membrane 22. The solution received by the membrane 22 from the projecting member 34 serves to maintain the pre-test metabolic condition of the cells. The solution may be, for example, water, saline, a growth medium, or oil for maintaining an aerobic condition.
In some embodiments, an excess quantity of the solution is drained from the projecting member 34 and the membrane 22. For example, the projecting member 34 may include a socket or slit into which the excess quantity of the solution is discharged. By discharging the excess quantity of the solution, such embodiments can avoid distortion of the image caused by having a greater quantity of solution than the membrane 22 can receive.
Further, in some embodiments, the membrane 22 is completely immersed in the solution. In some embodiments, the membrane 22 is saturated by and immersed in an oil-based solution especially conductive to the growth of anaerobic bacteria. In some embodiments, the membrane 22 merely touches the solution without being immersed in the solution. In certain embodiments, the solution can diffuse gradually through the membrane 22. In other embodiments, the solution may rise up from the projecting member 34 so as to contact the membrane 22 via capillary action.
Referring again to
The components of the apparatus 100 (and a system 200 described below) may be made out of any suitable materials and structures. For example, in some embodiments, the holder 20 is made out of a durable plastic. In some embodiments, the projecting member 34 is composed of a rigid outer material and a porous spongiform inner material. In other embodiments, the projecting member 34 is substantially solid. Suitable materials include durable plastics, glass and metals that can be readily cleaned.
With reference to
Referring again to
Still referring to
Turning now to
Referring now to
Referring once more to
Further, in some embodiments, the membrane 22 can be anchored to the projecting member 34 so as to be secured in place. The projecting member 34 thus provides a mechanical support to the membrane 22 so that deformation of the membrane 22 (beyond any deformation incurred in securing the membrane 22) can be avoided. By securing the membrane 22 in this manner, image acquisition can be carried out with high repeatability and improved focusing.
Referring now to
As shown in
Next, at 606, the process includes applying a solution to an apparatus for imaging such as the apparatus 100 described above. More particularly, a solution can be applied to a top surface of a projecting member such as the projecting member 34 via a dropper, a reservoir, or other solution-delivering mechanisms. Following the application of solution at 606, an excess of solution can optionally be drained at 608.
The process 600 for prolonged live cell imaging includes arranging the membrane 22 with the holder 20. The arranging of the membrane 22 and the holder 20 may take place at the beginning of process 600, before the sample is filtered through the membrane. As described above, the membrane 22 can be stretched by the holder 20 so as to be substantially straight and not susceptible to further deformation. The holder 20 containing the membrane 22 is positioned with the projecting member 34. More particularly, the membrane 22 is positioned such that the solution previously applied on the projecting member 34 contacts the membrane 22. Furthermore, at 610, the projecting member 34 is disposed so as to mechanically support the membrane 22 and to be anchored to it. In this manner, the projecting member 34 can protect the membrane 34 from deformation. Following anchoring of the membrane 22 to the projecting member 34, images of the cells can be carried out at 612.
Referring again to
Once more referring to
A process for determining a number of culturable microbial cells according to one embodiment is described below. The process begins with (1) arranging a membrane in a membrane holder, (2) filtering a tested sample through the membrane as to capture all the bacteria from the tested sample onto the filter. The process further involves (3) treating the bacteria according to the desired test protocol, such as dying the bacteria with a fluorescent dye, washing the cells or other preparatory processes that may be carried out so as to obtain desirable environmental and/or sampling conditions, for example. Next, the process involves (4) applying an appropriate solution to the upper surface of the stage protrusion, either by manually placing a drop, or by using a reservoir of solution, and (5) optionally draining excess solution to prevent image distortion. The process further entails (6) placing the filter on the stage protrusion such that the filter holder is anchored to the protrusion. The filter is placed to be in close proximity to the upper surface of the protrusion, and the filter is in touch with the solution and mechanically supported by the upper surface of the stage protrusion so as to prevent filter deformation and to enhance focus. The process further includes (7) acquiring images according to a desirable protocol.
It should be noted that the sequence of processes illustrated in
An alternative process of some embodiments includes, for example, binding cells from a sample to a membrane or filter such as the membrane 22, fixing the membrane 22 to a holder such as the holder 20, and applying solution to a stage protrusion such as the protrusion 34. The process can further include draining an excess amount of the solution applied to the stage protrusion before arranging the membrane 22 and the holder 20 on the stage protrusion 34.
In some embodiments, by way of example, the method can further include providing a glass cover slip atop the membrane 22 after the membrane 22 is secured in the holder 20. In some embodiments, the method can include staining the cells bound to the membrane 22 prior to fixing the membrane 22 to the holder 20. In some embodiments, the method includes mounting the membrane 22 to the holder 20, and subsequently staining cells bound to the membrane 22. In some embodiments, the cells are bound to the membrane 22, which is then fixed to the holder 20 and positioned to contact the projecting member 34 prior to staining.
In some embodiments, following arrangement of the membrane 22 and the holder 20, the cells on the membrane 22 are suspended with a substance such as a fluorescent dye, for example. After the staining, the cells are washed so as to reduce an amount of dye present in a background image to be taken of the dyed cells. Once the cells are washed, the cells are mounted to a stage such as the stage 32. It should be noted that the suspending and washing processes are illustrative of an exemplary preparatory process prior to imaging of the cells and may be omitted in some embodiments. Other preparatory processes may be carried out so as to obtain desirable environmental and/or sampling conditions for the cell to be imaged by the imaging device.
In some embodiments, the washing and staining of microbial cells can be performed before the cells are bound to the membrane, or can be performed on the membrane. The staining on the membrane can be before or after mounting the membrane 22 in the holder 20. In some embodiments, the membrane is assembled prior to filtering because the assembled membrane fits the filtering machine and thus goes directly from filtering to staining and imaging. When washing and staining is performed after mounting the member in the holder, the remaining features of the apparatus are suitably configured for the exchange of solutions with the membrane, such as by provision of tubes to provide for the exchange of solutions. Performing the staining and washing on a membrane 22 in the holder 20 may be an element of an automated process, e.g. to examine multiple samples.
In some embodiments, once the cells are mounted to the stage, the cells can undergo imaging by the imaging device. The imaging device thus allows images of the cells to be acquired. In some embodiments, the imaging device allows prolonged imaging of live cells so as to keep the cells alive and metabolically intact during imaging. For example, in some embodiments, live cells are imaged so as to determine the quantity of culturable cells from samples including dead cells and VNCs. Such embodiments can differentiate between viable culturable cells (VCC) and VNCs.
To obtain the images of
In particular,
In contrast to
E. Coli
E. Coli
The results shown in Table 1 demonstrate that without maintaining the membranes in a wet state, the membranes dry very fast and the cells change their metabolic state, and may lose their culturability, dry out and die.
Thus, the present device, composition and related method permit accurate enumeration of VCC.
The present invention is not limited to the above-described preferred embodiments. Various alternatives, modifications and equivalents may be used. Therefore, the above embodiments should not be taken as limiting the scope of the invention.
The present application claims priority to U.S. Provisional Application No. 61/936,725, filed Feb. 6, 2014, and U.S. patent application Ser. No. 14/271,190, filed May 6, 2014. The disclosures of these prior applications are incorporated by reference herein in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61936725 | Feb 2014 | US | |
| 61936725 | Feb 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15115248 | Jul 2016 | US |
| Child | 16153445 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14271190 | May 2014 | US |
| Child | 15115248 | US |
