Method for detecting, identifying, or counting microorganisms, and system using same

Abstract

The present invention relates to a method capable of detecting, identifying or counting microorganisms, and a system using the same, and provides a method capable of identifying, detecting or counting microorganisms in a more rapid, accurate and convenient manner than a conventional method for identifying, detecting or counting microorganisms. According to the present invention, it was found that identification, detection or counting of microorganisms can be performed in a rapid, accurate and convenient manner, when a fluorescently labeled microorganism sample is centrifuged and attached to the surface of a slide, followed by analysis of fluorescent images. Therefore, the method and system of the present invention can be useful in various fields requiring detection, identification and counting of microorganisms.

Claims

1. A device for detecting, identifying or counting microorganisms, comprising: (a) a slide having disposed on a surface thereof a metering chamber capable of receiving a sample solution containing microorganisms, wherein the metering chamber comprises an upper plate and a lower plate, and a chamber interior formed by coupling of the upper plate with the lower plate has a depth of 1 to 200 m and (b) a centrifugal means configured to apply a centrifugal force in a direction perpendicular to the lower plate of the metering chamber containing the sample solution, thereby forming a focal plane in the metering chamber; (c) a light source configured to irradiate light onto the slide; (d) an imaging means configured to acquire an image produced by the light source; (e) an image processor configured to detect, identify or count microorganisms in a predetermined volume of the metering chamber based on the image acquired by the imaging means; and (f) a display means configured to display the results of detecting, identifying or counting the microorganisms.

2. The device of claim 1, further comprising a fluorescent or luminescent substance capable of labeling the microorganisms.

3. The device of claim 1, further comprising a loading buffer which is used to apply the sample solution to the metering chamber.

4. The device of claim 3, wherein the loading buffer comprises a substance having a higher boiling point than water.

5. The device of claim 4, wherein the substance is one or more of glycerol, DMSO, fructose syrup, and polyethylene glycol.

6. The device of claim 1, wherein the metering chamber is a chamber capable of controlling volume of the solution that is received in the chamber.

7. The device of claim 1, wherein the focal plane is formed by a process in which the microorganisms contained in the sample solution loaded into the metering chamber form a monolayer on the lower surface of the metering chamber.

8. The device of claim 1, wherein two or more of the detecting, identifying and counting of the microorganisms are capable of being performed at the same time.

9. A method for detecting, identifying or counting microorganisms using the device of claim 1, comprising the steps of: (i) contacting a sample solution containing microorganisms with a fluorescent or luminescent substance capable of labeling the microorganisms; (ii) loading the sample solution, contacted with the fluorescent or luminescent substance, into the metering chamber disposed on the surface of the slide; (iii) centrifuging the slide, having the metering chamber disposed thereon, by the centrifugal means; (iv) acquiring a fluorescent or luminescent image from the metering chamber, disposed on the slide surface, by the imaging means; and (v) analyzing the fluorescent or luminescent image, thereby detecting, identifying or counting the microorganisms contained in the sample solution.

10. The method of claim 9, wherein the fluorescent or luminescent substance in step (i) labels nucleic acids of the microorganisms.

11. The method of claim 9, wherein the fluorescent or luminescent substance in step (i) labels activity of viable microorganisms.

12. The method of claim 9, wherein the fluorescent or luminescent substance in step (i) is a substance conjugated to an antibody.

13. The method of claim 12, wherein the antibody labels a microorganism-specific antigen.

14. The method of claim 9, wherein the centrifuging in step (iii) is performed such that a centrifugal force acts in a direction perpendicular to the lower plate of the metering chamber loaded with the sample solution, thereby forming a focal plane.

15. The method of claim 14, wherein the focal plane is formed by a process in which the microorganisms contained in the sample solution loaded into the metering chamber form a monolayer on the lower surface of the metering chamber.

16. The method of claim 9, further comprising, after step (i), a step of mixing the sample solution, contacted with the fluorescent or luminescent substance in step (i), with a loading buffer, and step (ii) comprises loading the mixture of the loading buffer with the sample solution contacted with the fluorescent or luminescent substance, in place of the sample solution contacted with the fluorescent or luminescent substance in step (i), into the metering chamber disposed on the slide surface.

17. The method of claim 16, wherein the loading buffer comprises a substance having a higher boiling point than water.

18. The method of claim 17, wherein the substance is one or more of glycerol, DMSO, fructose syrup, and polyethylene glycol.

19. The method of claim 9, wherein two or more of the detecting, identifying and counting of the microorganisms are capable of being performed at the same time.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a schematic view showing the method of the present invention.

(2) FIG. 2 depicts photographs showing the results of fluorescently imaging various kinds of microorganisms stained with a nucleic acid-staining fluorescent dye.

(3) FIG. 3 depicts photographs showing the results of measuring the time-dependent evaporation of a solution loaded into a metering chamber slide.

(4) FIG. 4 depicts photographs showing the results of imaging cells without centrifugation, in comparison with the results of imaging cells after centrifugation.

(5) FIG. 5 shows the results obtained by loading each of 1:10, 1:20 and 1:100 of E. coli into a 100-m chamber slide, centrifuging the loaded E. coli, and observing the centrifuged E. coli.

(6) FIG. 6 shows the results obtained by loading each of 1:10, 1:20 and 1:100 of E. coli into a 100-m chamber slide, and observing the loaded E. coli without centrifugation.

(7) FIG. 7 is a schematic view illustrating a cell imaging system.

(8) FIG. 8 depicts photographs showing the imaging results acquired by a cell imaging system having a plurality of fields of view.

(9) FIG. 9 is a table comparing the results of counting bacteria by the method of the present invention with the results of counting bacteria by a spread plate method.

BEST MODE

(10) Hereinafter, a system for identifying, detecting or counting microorganisms according to the present invention, and a system using the same, will be described in detail with reference to the accompanying drawings. The examples described below are for illustrative purposes only and are not intended to limit the scope of the present invention.

EXAMPLES

Example 1

Labeling of Microorganisms with Fluorescent Substance

(11) In order to label various microorganisms including E. coli, microorganisms were stained with the nucleic acid-staining dye MycoLight (ATT Bioquest, USA). Specifically, microorganisms were diluted and suspended in Dulbecco's phosphate-buffered saline (DPBS) solution at various ratios (such as 1:2 to 1:100), and the suspension of the microorganisms was brought into contact with MycoLight and allowed to react for 5 to 30 minutes so that DNA of the microorganisms would be labeled with MycoLight. As the reaction buffer, 2.5PET (2.5 mM EDTA and 0.025% Tween-20 in 2.5PBS) or 1DPBS was used. FIG. 2 shows fluorescence images of various microorganisms fluorescently labeled by contact with MycoLight as described above.

Example 2

Determination of Composition of Loading Buffer

(12) The amount of sample loaded into the metering chamber slide is a very small (several tens of l or less), and for this reason, a separate loading buffer is required to inhibit phase change resulting from sample evaporation or a change in external pressure after loading of the sample into the metering chamber. Furthermore, the loading buffer is required so that a particular focal plane formed after centrifugation of the metering chamber slide can be maintained without being deformed by physical impact. For such purposes, an experiment was performed in order to use glycerol (Samchun Pure Chemical Co., Ltd., Korea), DMSO (Sigma, USA) or X-clarity mounting solution (hereinafter referred to as M/S; Logos Biosystems, Inc.), which has a higher viscosity and boiling point than aqueous solution. FIG. 3 shows the results obtained by loading solutions containing various concentrations of glycerol into the 100-m-depth chamber of a metering chamber slide, and then observing the time-dependent evaporation of the solutions while incubating the solutions at room temperature. In order to easily observe the degree of evaporation of the solutions, trypan blue dye (Life Technologies, USA) together with glycerol was added to the loaded solutions. As shown in FIG. 3, the degree of evaporation of the solutions in the chamber significantly decreased as the concentration of glycerol increased. DMSO having a higher boiling point than water also showed a similar effect. As described above, the condition where evaporation of the solution in the chamber is prevented by an additive having a high boiling point significantly inhibited the evaporation of a sample, which would occur either during sample handling before or after concentration or during centrifugation. The experimental results indicated that 80 v/v % M/S or 40 v/v % glycerol (final concentration) can be used as a loading buffer and that it is enough to perform centrifugation at 100 RCF for 10 minutes

Example 3

Centrifugation of Slide

(13) In order to centrifuge the slide so as to effectively form a focal plane, the kind and concentration of loading buffer added to a sample, the posture of the metering chamber slide during centrifugation, rotating speed and time during centrifugation, etc., were examined. FIG. 4 shows images obtained by staining E. coli DH5 cells with a nucleic acid-staining MycoLight reagent, mixing the cells with a loading buffer (glycerol), loading 2.5 l of the mixture into the metering chamber, and then fluorescently imaging the centrifuged slide and the non-centrifuged slide for comparison. Centrifugation was performed at 500 RCF for 30 seconds. In centrifugation, a centrifugal force acted in a direction perpendicular to the bottom of the metering chamber slide. The slide was imaged and analyzed using a digital fluorescence microscope (iRiS (trade name), Logos Biosystems, Inc.) equipped with a 10 objective lens, and as a result, it was shown that, when centrifugation was not performed, the cells were not on the same focal plane, and for this reason, all the cells could not be imaged on a specific focal plane, and a large number of the cells were imaged in an unfocused state. However, when centrifugation was performed, almost all of the cells were on a single focal plane, and almost all of the cells could be imaged on a specific focal plane.

(14) After changing the chamber slide depth (20 m and 100 m), the kind of loading buffer (PBS and glycerol) and centrifugal speed (40 RCF and 900 RCF), cell deflection, focal plane formation, focal plane deviation, and cell movement were observed. The following buffer solutions were prepared and used: a buffer solution obtained by mixing 20 l of E. coli, 4 l of MycoLight and 16 l of 2.5PET and adding 40 l of 80% glycerol to the mixture; and a buffer solution obtained by mixing 20 l of E. coli, 4 l of MycoLight and 56 l of DPBS. When the slide depth was low, sample aggregation appeared after centrifugation, and in the conditions of 100-m chamber slide depth, 40 RCF, and glycerol, about 80% of E. coli cells in the sample precipitated, and large focal plane derivation (32 m) was formed. PBS started to dry on a 20-m slide before centrifugation. The results are shown in Table 1 below.

(15) The sample obtained by adding and reacting glycerol as described above was transferred to a 100-m-depth chamber slide, centrifuged at each of 50, 100, 200 and 400 RCF for 10 minutes, and then analyzed. The results of the analysis are shown in Table 2 below. 90% or more of the sample precipitated at 100 RCF or higher, and deflection at the inlet started to appear from 100 RCF.

(16) TABLE-US-00001 TABLE 1 Centrifuge speed Slide Loading 40 RCF 900 RCF depth buffer PBS Glycerol PBS Glycerol 20 m Deflection Moderate None Occurred Occurred Focal plane None None None None deviation Sample Moderate None Moderate None movement 100 m Deflection Moderate None Occurred Occurred Focal plane None Occurred None None deviation Sample Occurred None Moderate None movement

(17) TABLE-US-00002 TABLE 2 Distribution deviation Focal plane between the deviation (Z- Deflection Deflection center and RCF axis) (inlet) (outlet) the edge 0 91.2 m 50 55.13 m None Slight None 100 18.97 m Slight Occurred Slight 200 9.23 m Occurred Occurred Occurred 400 8.35 m Occurred Occurred Occurred

(18) A 100-m-depth chamber slide, a staining reagent (AAT Bioquest Cat #24001, Lot #106137) comprising a solution of 200 M of MycoLight in DMSO, 2.5reaction buffer (2.5PET) (a solution of 2.5 mM EDTA and 0.025% Tween-20 in 2.5PBS), 80% glycerol, and iRiS TC PlanFluor (Logos Biosystems, Inc.) were used. E. coli was diluted in DPBS at ratios of 1:10, 1:20 and 1:100. 10 m microorganisms, 2 l of staining reagent, and 2.5PET were reacted. After 30 minutes of the reaction, 20 l of loading buffer was added to the reaction mixture. The prepared sample was transferred to a chamber slide, and then the sample before centrifugation was compared with the sample after centrifugation. The results are shown in FIGS. 5 and 6. It could be seen that, in comparison with an image obtained without centrifugation (FIG. 6), the focal plane in an image obtained after centrifugation (FIG. 5) was uniformly formed. In addition, deflection after centrifugation was examined at varying centrifugal speeds (200, 400, 600 and 800 RCF). Furthermore, precipitation of microorganisms in the sample was analyzed while reducing the centrifugal speed. E. coli diluted at a ratio of 1:10 as described above was used, and the chamber slide was centrifuged at each of 40, 50 and 100 RCF for 10 minutes, and the results were compared. Table 3 below shows focal plane deviation.

(19) TABLE-US-00003 TABLE 3 RCF Focal plane deviation (Z-axis) 40 9.18 m 50 7.78 m 100 4.43 m

Example 4

Acquisition and Analysis of Fluorescent Images

(20) In order to acquire fluorescent images, an automated imaging system as shown in FIG. 7 was constructed. In order to image the fields at various positions in a single metering chamber slide, automated X-Y stages were constructed, each comprising a single LED, an excitation filter and an emission filter. Using this automated imaging system, images of the slide centrifuged according to the method of Example 3 were acquired (FIG. 8). The automatically acquired images were processed by image analysis software (Logos Biosystems, Inc., Korea), and then the number of particles emitting a fluorescent signal was counted, thereby determining the number of microorganisms per unit volume. The results of counting microorganisms by the method of the present invention and the results of counting microorganisms by a conventional spread plate method were comparatively analyzed (FIG. 9). In FIG. 9, DC represents the results obtained by converting the number of cells, counted using image analysis software, into concentration per unit volume (ml), and CFU (colony forming unit) represents the results obtained by converting the number of colonies, formed in spread plate culture, into the concentration of cells capable of forming colonies per unit volume (mL). Corr (correlation) is a value obtained by dividing DC (D) by CFU (C). The results of the analysis indicated that the method for counting microorganisms according to the present invention has a high correlation with the method of counting microorganisms by the spread plate method. Although there was a difference depending on the kind of loading buffer, a dye gradient was formed when the depth of the chamber on the slide was low.

(21) Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

INDUSTRIAL APPLICABILITY

(22) As described above, the present invention can be widely used for the detection, counting and identification of small microorganisms such as bacteria, and thus can be used for the detection, counting and identification of microorganisms in various fields.