Device and method for determining the action of active ingredients on nematodes and other organisms in aqueous tests

Abstract

The invention relates to a device (1) and a method for determining the action of active ingredients on nematodes and other organisms in aqueous tests. The device (1) according to the invention comprises a holder (13) for a cell culture plate (30) having multiple wells (31) in which the nematodes can be filled with the active ingredients, said cell culture plate (30) having a bottom side (33), a top side (32) and also side walls extending between bottom side (33) and top side (32), a camera (11) which is used to record images of preferably the bottom side (33) of the cell culture plate (30), a lighting mechanism (14) having at least a first light source (15) which illuminates the cell culture plate (30), there being arranged between the first light source (15) and a first side wall (34) of the cell culture plate (30) in the installed state a first optical unit which directs the light of the first light source (15) through the first side wall (34) in the direction of the bottom side (33) of the cell culture plate (30). The method according to the invention makes it possible to simultaneously investigate many active ingredients within a very short time.

Claims

1. Device for determining the action of active ingredients on nematodes and other organisms in aqueous tests, comprising a holder for a cell culture plate having multiple wells in which the nematodes can be filled with the active ingredients, said cell culture plate having a bottom side, a top side and also side walls extending between bottom side and top side; a camera which is used to record images of preferably the bottom side of the cell culture plate; a lighting mechanism having at least a first light source which illuminates the cell culture plate; there being arranged between the first light source and a first side wall of the cell culture plate in the installed state a first optical unit which directs the light of the first light source-through the first side wall in the direction of the bottom side of the cell culture plate.

2. Device according to claim 1, wherein the optical unit has a rod lens which substantially spans the entire length of the first side wall of the cell culture plate.

3. Device according to claim 1, wherein the first light source has a line light guide.

4. Device according to claim 1, wherein the optical unit prevents a direct illumination of a top well-cover of the cell culture plate.

5. Device according to claim 1, wherein the optical unit prevents a direct penetration of light into an objective of the camera.

6. Device according to claim 1, wherein the plate holder is composed of transparent material.

7. Device according to claim 2, wherein a gap between first light source and rod lens is between 2 and 4 cm.

8. Device according to claim 1, wherein the lighting mechanism has a second light source and a second optical unit, which is arranged on a second side wall of the cell culture plate, said second side wall being opposite to the first side wall.

9. Method for determining the action of active ingredients on nematodes and other organisms in aqueous tests, comprising: a) filling at least one well of a cell culture plate with nematodes and an active ingredient; b) arranging the cell culture plate in the device according to claim 1; c) creating multiple images of the cell culture plate that follow one another chronologically; d) binarizing the created images depending on an illumination of the cell culture plate; e) determining a first measurement curve based on the well for a first series of images, said first series having a base image and multiple follow-up images, with each follow-up image being compared to the base image in a difference method and a number of difference pixels being determined; f) determining at least a second measurement curve based on the well for a second series of images, and, for the second series, using a follow-up image of the first series as base image and at least one further follow-up image of the first series as a follow-up image of the second series; g) determining an averaged curve on the basis of the first measurement curve and of the at least second measurement curve.

10. Method according to claim 9, wherein what is used for the base image of the second series is a first follow-up image of the first series, which first follow-up image immediately follows the base image of the first series, in that what are used for the second series are all follow-up images of the first series as follow-up images of the second series, except for the first follow-up image of the first series, and in that the second series is completed by an additional follow-up image.

11. Method according to claim 9, wherein a recording period (T.sub.A) between the base image and the last follow-up image of a series is established such that, within said recording period (T.sub.A), an asymptotic limit AW is reached for the number of difference pixels for a well in which untreated nematodes are situated.

12. Method according to claim 11, wherein a total period is established between the base image of the first series and a last follow-up image of a last series, which total period approximately corresponds to twice that of a recording period (T.sub.A).

13. Method according to claim 9, wherein the time interval between two successive images is constant and is from 1 to 5 seconds.

14. Method according to claim 9, wherein one series of images comprises from 8 to 12 images.

15. Method according to claim 10, wherein the averaged curve is determined on the basis of from 8 to 12 measurement curves.

Description

[0028] The invention shall be more particularly elucidated on the basis of the exemplary embodiments depicted in the figures, in which:

[0029] FIG. 1 shows schematically in cross section one exemplary embodiment of the device according to the invention;

[0030] FIG. 2 shows schematically the arrangement of a cell culture plate and a lighting mechanism of the device according to FIG. 1;

[0031] FIG. 3 shows a flow chart of one exemplary embodiment of the method according to the invention; and

[0032] FIG. 4 shows a measurement curve for a well containing untreated nematodes and a measurement curve for nematodes treated with an active ingredient.

[0033] FIG. 1 shows schematically in cross section a device for determining the action of active ingredients on nematodes. The device 1 has a housing 10, in which a camera 11 having an objective 12 is arranged. Furthermore, the housing 10 is provided with a holder 13 for a cell culture plate 30 which comprises multiple wells 31. The cell culture plate 30 has a top side 32 and a bottom side 33. Arranged between the top side 32 and the bottom side 33 of the rectangular cell culture plate 30 are four side walls, of which a first side wall 34 and an opposing second side wall 35 can be recognized in the depiction in FIG. 1.

[0034] A lighting mechanism 14 having a first light source 15 and a second light source 16 is likewise arranged in the housing 10. Arranged between the first light source 15 and the first side wall 34 is, as part of a first optical unit, a rod lens 17, which, like the first light source 15, spans the entire length of the first side wall 34. A rod lens 18 is arranged too between the second light source 16 and the second side wall 35.

[0035] The device 10 makes it possible, using the camera 11, to create multiple digital images of the cell culture plate 30 that follow one another chronologically, the images being recorded from the bottom side 33 of the cell culture plate 30. Accordingly, the camera 11 with its objective 12 is arranged below the holder 13 for the cell culture plate 30. Here, the device 10 has means, which are not further depicted, for storing and for processing the images recorded by the camera 11. Alternatively, the device 10 can be connected to appropriate (computer) means.

[0036] FIG. 2 shows, on an enlarged scale, the arrangement of the cell culture plate 30 and the lighting mechanism 14 having the first light source 15 and the second light source 16. The individual wells 31 of the cell culture plate 30 are filled with an aqueous solution 36 in which from 50 to 100 nematodes and an active ingredient to be investigated are situated. FIG. 2 depicts eight wells 31 arranged in a row, and twelve rows arranged next to one another would give altogether 96 individual wells 31. Other patterns for the cell culture plate 30 are possible, for example a 4×6 pattern or 6×8 pattern.

[0037] Different active ingredients can be filled into the different wells 31. In addition, there can also be wells in which only nematodes without active ingredient are situated in the aqueous solution.

[0038] The rod lenses 17, 18 cause the light of the light sources 15, 16 to be directed in the direction of the bottom side 33. At the same time, the rod lenses 17, 18 prevent the light of the light sources 15, 16 from directly reaching the top side 32 of the cell culture plate 30, there being provided on the top side 32 a film 37 which covers the individual wells 31 from above. In addition, the rod lenses 17, 18 or the arrangement of the rod lenses 17, 18 are designed such that no light directly falls into the objective 12 of the camera 11. 17a and 17b indicate light beams emerging from the rod lens 17. Corresponding exit beams of the rod lens 18 are indicated by 18a, 18b.

[0039] FIG. 2 reveals that the rod lenses 17, 18, at least the central axes thereof extending perpendicularly to the drawing plane, are arranged between the top side 32 and the bottom side 33 of the cell culture plate 30 when the cell culture plate 30 is situated in the holder 13 of the device 10, which holder 13 is intended for said cell culture plate 30.

[0040] FIG. 3 shows a flow chart of one exemplary embodiment of the method according to the invention. The flow chart starts with a start block 100. In block 101, an asymptotic value AW is determined on the basis of untreated nematodes. In this connection, the asymptotic value AW is the number of difference pixels which can maximally arise when a follow-up image is compared with a base image. FIG. 4 shows such an asymptotic value AW for a measurement curve 50 for untreated nematodes. In this connection, the individual measurement points reflect the number of difference pixels arising in the comparison of the follow-up images recorded at different times with a base image recorded at time t=0. In this connection, the asymptotic value AW is linked to a recording period T.sub.A, within which the measurement curve 50 at least approximately reaches the asymptotic limit AW. In the present example, the recording period shall be 27 seconds, and the interval between two adjacent follow-up images or the interval between the first follow-up image and the base image (t=0) shall be three seconds. Therefore, a number n.sub.AW of images per measurement curve including the base image (t=0) is equal to 10.

[0041] FIG. 4 also shows exemplarily an (averaged) measurement curve 51 for treated nematodes. What can be seen is that the measurement curve 51 runs below the measurement curve 50 for the untreated nematodes, since the treated nematodes move more slowly or some of them no longer move. The smaller the area under the measurement curve 51 with regard to the area below the measurement curve for untreated nematodes, the stronger the action of the corresponding active ingredient. The averaged measurement curve 51 will, however, be discussed in more detail later on.

[0042] After the determination of the asymptotic value AW on the basis of untreated nematodes and after the establishment of the number of images n.sub.AW per measurement curve or per series (block 101), what then takes place is, according to FIG. 3 in block 102, the recording of a certain number of images Image_1 to Image_x. At this point, it should be pointed out that the camera 11 creates at each time a total image of the cell culture plate 30 with all wells, and the images Image_1 to Image_x are then cut out or created for each individual well from said total image in a well-based manner. Said images are then binarized in block 103. In the binarization, the individual pixels are assigned either to the nematodes (white pixel) or to the background (black pixel). The binarization in block 103 must take place downstream of an establishment of a threshold value for the intensity of the pixel, which value makes it possible to divide the pixels into “white” and “black”.

[0043] For a first measurement curve m=1 (see block 104), the images Image_2 to Image_n.sub.AW are then used, in that the number of difference pixels between initially a first follow-up image Image_2 and the base image Image_1 is determined by means of a difference method (see block 105 and block 106). In this connection, block 106 is passed through multiple times as part of a loop, and so the number of difference pixels is determined for multiple follow-up images (Image_2-Image_1;-Image_3-Image_1;-Image_4-Image_1; . . . ; Image_n.sub.AW-Image_1). The loop 107 is exited when the number of images for the first measurement curve has reached the value n=n.sub.AW+m−1 and the query 108 provided within the loop 107 cannot be answered with “Yes”. In this case, all values are available for creating the first measurement curve m=1 (cf. block 109).

[0044] After creation of the first measurement curve m=1, the loop 110 is then used to create further measurement curves (m=2, m=3, . . . , m=m.sub.max). In this connection, a second measurement curve is based on the difference images Image_3-Image_2;-Image_4-Image_2; -Image_n.sub.AW+1-Image_2. Therefore, Image_3, for example, is used both for the creation of the first measurement curve m=1 and for the second measurement curve m=2.

[0045] When a query 111 within the loop 110 cannot be answered with “Yes”, the loop 110 is exited. All measurement curves 1, 2, . . . , m.sub.max are now available, and so these measurement series can be averaged in block 112. It has been found that this averaging of the individual measurement series, which rely on the same images for the most part, can considerably reduce the statistical scattering.

[0046] Thus, it is possible using altogether m+n.sub.AW-1 images to generate m measurement series, each containing n.sub.Aw measurement points (including the zero point).

[0047] In block 113, the integral of the averaged curve is calculated, and said integral can then be compared with the area under the measurement curve 50 for untreated nematodes (see block 114).

[0048] Assuming that the measurement series 51 already mentioned above in FIG. 4 corresponds to the averaged measurement series according to block 112 and that the area below the measurement curve 50 comprises I.sub.50=100 area units and the area below the (averaged) measurement curve comprises I.sub.51=65 area units, it is possible to provide information about the efficacy of the active ingredient in accordance with the following formula: (I.sub.50(untreated)-I.sub.51(treated))/I.sub.50(untreated)100%. In the case of the number example taken as a basis here, a value of 35% would then arise.

[0049] Thus, it is, for example, possible in the case of a cell culture plate having 96 wells to investigate almost 100 active ingredients at the same time. 60 seconds are sometimes enough for the recording of the images required for generating a sufficient number of measurement series with sufficient measurement points in order to achieve statistically reliable results. The invention therefore allows a rapid and efficient investigation of the efficacy of active ingredients on nematodes or similar organisms.

List of Reference Signs

[0050] 1 Device

[0051] 10 Housing

[0052] 11 Camera

[0053] 12 Objective

[0054] 13 Holder

[0055] 14 Lighting mechanism

[0056] 15 First light source

[0057] 16 Second light source

[0058] 17 Rod lens (17a, 17b exit beams)

[0059] 18 Rod lens (18a, 18b exit beams)

[0060] 30 Cell culture plate

[0061] 31 Well

[0062] 32 Top side

[0063] 33 Bottom side

[0064] 34 First side wall

[0065] 35 Second side wall

[0066] 36 Solution

[0067] 37 Well-cover

[0068] 100 Start block

[0069] 101 Block

[0070] 102 Block

[0071] 103 Block

[0072] 104 Block

[0073] 105 Block

[0074] 106 Block

[0075] 107 Loop

[0076] 108 Query

[0077] 109 Block

[0078] 110 Loop

[0079] 111 Query

[0080] 112 Block

[0081] 113 Block

[0082] 114 Block