AUTOMATIC METHOD FOR MONITORING CELL CULTURE GROWTH

Abstract

In the automated method according to the invention for monitoring cell culture growth, in particular bacterial growth, a receiving dish (24) with a nutrient medium (38) is provided onto and/or into which is applied a cell culture, in particular a sample of human or animal tissue, such as blood, to which bacteria have been added and which also contains one or a plurality of reagents, in particular antibiotics. A microscope (10) having a camera (18) and an image acquisition plane adapted to be moved along the optical axis (20) is provided, and the receiving dish (24) is brought into the microscope (10) for monitoring a potential growth of the cell culture in the nutrient medium (38). At predetermined time intervals, in particular in the single-digit minute range, an image of the nutrient medium (38) is acquired using the camera (18) of the microscope (10) by moving the image acquisition plane along the optical axis (20) through the nutrient medium (38) by means of the movable slide (14) and/or the acquisition optic unit (16), one image per image acquisition plane is acquired, and from the group of acquired images the highest-contrast image of the nutrient medium (38) or an image of the nutrient medium (38) with a contrast sufficient for the subsequent automatic further processing of the image is automatically selected and stored, where required, by means of an image evaluation software. By means of the image evaluation software the size of the area occupied by the cell culture is automatically determined on the basis of the selected image. On the basis of the sizes of the areas occupied by the cell culture of the respective selected images it is determined whether the cell culture is growing or not.

Claims

1. An automated method for monitoring cell culture growth, in particular bacterial growth, wherein in the method comprises: providing a receiving dish with a nutrient medium onto and/or into which is applied a cell culture, in particular a sample of human or animal tissue, such as blood, to which bacteria have been added and which also contains one or a plurality of reagents, in particular antibiotics, providing a microscope having an optical axis and including a camera and a slide for said receiving dish adapted to be automatically moved along said optical axis and/or an acquisition optic unit adapted to be automatically moved along said optical axis, wherein said receiving dish is brought into said microscope for monitoring a potential growth of the cell culture in said nutrient medium, acquiring at predetermined time intervals, in particular in the single-digit minute range, an image of said nutrient medium is acquired using said camera of said microscope by moving the image acquisition plane along said optical axis through said nutrient medium by means of at least one of said movable slide and said acquisition optic unit of said microscope, one image per image acquisition plane is acquired, and from the group of acquired images the highest-contrast image of said nutrient medium or an image of said nutrient medium with a contrast sufficient for the subsequent automatic further processing of the image is automatically selected and stored, where required, by means of an image evaluation software, wherein by means of the image evaluation software the size of the area occupied by the cell culture is automatically determined on the basis of the selected image, and wherein on the basis of the sizes of the areas occupied by the cell culture of the respective selected images it is determined whether the cell culture is growing or not.

2. The method according to claim 1, wherein on the basis of the sizes of the areas occupied by the cell culture of the respective selected images it is determined whether the cell culture is growing, and if so, at which growth rate.

3. The method according to claim 1, wherein the step of the image acquisition plane traveling through the receiving dish and the nutrient medium is effected by moving optical elements of the acquisition optic unit of the microscope along the optical axis and/or by moving the slide comprising said receiving dish along said optical axis of said microscope.

4. The method according to claim 1, wherein the region within which the image acquisition plane travels in the nutrient medium for determining the image of said nutrient medium with the highest contrast or for determining an image of said nutrient medium with a contrast sufficient for determining the size of the area of the cell culture is limited to the region around the position of the image acquisition plane of the temporally last image or one of the temporally previous images.

5. The method according to claim 1, wherein the receiving dish comprises two sheets, in particular an object slide and a cover slip having a frame arranged therebetween, wherein the region surrounded by said frame defines the receiving space of said receiving dish.

6. The method according to claim 1, wherein on the basis of the sizes of the areas occupied by the cell culture of the respective selected images a growth curve is prepared.

7. The method according to claim 1, wherein for selecting the highest-contrast image or for selecting the image with a contrast sufficient for the subsequent further processing of the image either an image is first acquired and stored for each approached image acquisition plane and the selection is subsequently carried out, or the image acquired first is stored as the image determined as having the highest contrast so far and each further acquired image is compared to the stored image, and when this image has a higher contrast than the stored image, it is stored as the image determined as having the highest contrast so far.

8. The method according to claim 1, wherein the microscope is a reflected-light or a transmitted-light microscope.

Description

[0039] Hereunder the invention is described in detail with reference to the drawing in which

[0040] FIG. 1 schematically shows a representation of a reflected-light microscope to be used according to the invention,

[0041] FIG. 2 shows a magnified view of the object slide arrangement having a miniaturized receiving dish,

[0042] FIG. 3 schematically shows the functional principle for automatically determining the highest-contrast image,

[0043] FIG. 4 shows examples of pictures of the growing cell culture at various points in time A, B and C and the processed images corresponding to this pictures for foreground/background recognition for the purpose of automated determination of cell culture areas, and

[0044] FIG. 5 shows three examples of growth curves prepared according to the invention.

[0045] In FIG. 1 an exemplary embodiment of a reflected-light microscope 10 is shown which comprises a microscope body 12 having a reflected-light illumination unit 13 and a slide 14 (movable Z stage) adapted to be moved in particular in very small steps along the optical axis of the microscope. The microscope body 12 further includes an acquisition optic unit 16 and a camera 18. The optical axis is indicated at 20.

[0046] Alternatively to the reflected-light microscope the invention also allows for employing a transmitted-light microscope. The position of the transmitted-light illumination unit to be employed in this case is shown as a dashed line at 22.

[0047] On the slide 14 a receiving dish 24 is located which is shown in FIG. 2 at a larger scale. The receiving dish 24 comprises a bottom object slide 26 and a cover slip 28, in particular configured as two sheets or panes 30, 31 between which a frame 34 in particular configured as a double-faced adhesive film 32 is arranged. This frame 34 defines in its interior a receiving space 36 for a nutrient medium 38 (which is agar, for example) on which bacteria 40 grow. The nutrient medium is provided with a reagent, such as an antibiotic. The bacteria 40 may e. g. be part of a blood sample which is to be examined.

[0048] FIG. 3 illustrates the functional principle of the automated determination of the highest-contrast image which is acquired at a certain point in time after the start of the bacterial growth. By means of the slide 14 adapted to be moved in very small steps the image acquisition plane is moved through the receiving dish 24. In doing so, one image is acquired per step. The contrast intensity in different Z-positions is queried. In FIG. 3 this is shown using the example of a bacterial culture of Staphylococcus aureus. The graph of FIG. 3 shows the contrast intensity (in A. U.) as a function of the Z-position (in μm). Three images showing the associated contrast intensities are exemplarily shown.

[0049] FIG. 4 shows examples of the foreground/background recognition by means of the aforementioned ZETA software of the applicant using the example of an Escherichia Coli (E. coli) growth analysis. The upper row (A-C) shows the original images, wherein A represents the start of a time series, B has been acquired after approximately one hour and C has been acquired after 6 hours. The second lower row (A′-C′) shows the corresponding foreground/background recognition as black-and-white representations.

[0050] Finally, FIG. 5 shows three examples of bacterial growth curves determined according to the invention of a test for resistance to antibiotics of E. coli for LB medium (see curve 42), for ampicillin (see curve 44) and for kanamycin (see curve 46).