WO2000032743A1

WO2000032743A1 - Apparatus for automatic reading of antibiogram with improved contrast

Info

Publication number: WO2000032743A1
Application number: PCT/FR1999/002845
Authority: WO
Inventors: Michel Roch; Christian Curel
Original assignee: Intelligence Artificielle Applications Sarl
Priority date: 1998-11-30
Filing date: 1999-11-19
Publication date: 2000-06-08
Also published as: FR2786499A1; FR2786499B1

Abstract

The invention concerns an apparatus for automatic reading of an antibiogram, said apparatus comprising an illumination system (4) for illuminating a Petri dish (B) containing antibiotic lozenges positioned on a culture medium seeded with a germ, and an electronic CDD camera (1) for acquiring the antibiogram image. The invention is characterised in that it is equipped with an optical filter (3) adapted so that the camera (1) can acquire said image in a range of wavelengths determined by the maximum values of the ratio Q = Rs(μ)-Rg(μ)/Rs(μ) wherein Rs(μ) is the reflection factor of the germ-free culture medium, based on the wavelength μ and Rg(μ) is the reflection factor of the same medium, but seeded with germs.

Description

APPARATUS FOR AUTOMATICALLY READING AN ANTIBIOGRAM WITH IMPROVED CONTRAST

The present invention relates to an apparatus for automatically reading an antibiogram. In known manner, an antibiogram is carried out in the following manner to determine which antibiotics are capable of blocking the multiplication of a germ. A liquid sample containing the germ or strain of bacteria to be tested is carried out, Petri dishes containing different culture media are prepared and inoculated with the infectious liquid; then incubate and thus determine which culture media allowed the development of the germ, so which is the family to which the germs of the sample belong, after which, to go further in the identification, we takes up one or more colony (s) which are sown on culture media or in a gallery in order to find the species.

In parallel with this identification, an antibiogram is carried out by inoculating the germ on a Petri dish containing the appropriate agar, the inoculation being carried out regularly over the entire surface of the agar by any of the known techniques. We then place pellets of different antibiotics on the agar surface at regularly spaced points, it being understood that we choose a panel of antibiotics which we know is generally effective for germs of the determined family; the Petri dish is then placed in the oven and incubated for 18 hours. Germs develop on the agar surface except in areas where the antibiotic has blocked development; in this way around each pellet, a disc is formed in which the germ-free, that is to say shiny, agar is seen, while between the discs relating to the different pellets, the development of germs gives birth to a mat surface. The diameter of the outer circle of the disc relating to each tablet is noted and the minimum inhibitory concentration MIC for each antibiotic is calculated therefrom (the MIC is the minimum concentration of antibiotic necessary to inhibit the growth of a germ): plus the diameter the larger the circle, the more effective the antibiotic, and therefore the smaller the MIC. If, for a antibiotic, the MIC is too high, this means that the antibiotic is unusable; therefore below a certain diameter Dl of the inhibition circle, it is known that the antibiotic of the lozenge is unusable for the fight against the germ. Above a certain diameter D2, it is clear that the antibiotic is active in vitro. On the other hand, between the diameters Dl and D2, there is a zone of uncertainty.

There are already devices intended to perform the automatic reading of Petri dishes allowing the definition of antibiograms. These Petri dishes can be round or square. In current equipment, the Petri dish is placed in an area which is illuminated by means of a fluorescent tube of annular shape placed behind a cylindrical glass translucent allowing to have a substantially uniform illumination on the whole of the Petri dish which is placed in the center of said cylinder. The difficulty, when carrying out a computer acquisition of the image of the Petri dish, stems from the fact that it is necessary to have as strong contrasts as possible between the inhibition circle which surrounds the pellet of antibiotic, on the one hand, and the environment around this circle which corresponds to the area where the germ has grown. The image acquisition is, in known manner, made by means of a charge-coupled matrix electronic camera, called "CCD", the field of observation of which encompasses the entire Petri dish. There is often around the inhibition circle an annular transition zone whose appearance is intermediate between that of the inhibition circle and that of the environment where the germ has developed freely, and this transition zone has an aspect which varies radially continuously from one of the edges to the other edge of the annular transition zone. It is clear that this circumstance makes particularly difficult the computer determination, from the image captured by the CCD camera, of the diameter to be taken into consideration for the calculation of the MIC.

But regardless of this phenomenon, another circumstance sometimes makes it particularly difficult to read the image of. the Petri dish; this happens in particular when the agar on which the germ has developed is an opaque or dark-colored agar, for example a red medium based on sheep's blood or a medium brown based on heated blood, on which it is very difficult to see the contrast between the inhibition disc and the surrounding area; this happens in particular for agar supports containing blood.

Reading the antibiogram is also difficult when the growth of the germ on the culture medium is weak, which makes it difficult to distinguish the inhibition discs from the rest of the environment where the germ has grown freely.

Also known from US Pat. No. 4,701,850 is an antibiogram reading device comprising a diffuse lighting source to illuminate the surface of the Petri dish and an electronic or digital caliper to measure the diameter of the circles from a distance. inhibition around antibiotic lozenges. However, this device does not allow automatic reading, and also presents the aforementioned problems related to contrast. The object of the invention is to propose an automatic device for reading an antibiogram, which makes it possible to improve the contrasts on the surface of a Petri dish, to facilitate reading by a CCD camera.

To this end, the subject of the invention is an apparatus for automatic reading of an antibiogram, said apparatus comprising a lighting system for illuminating a Petri dish containing antibiotic pellets positioned on a culture medium seeded with a germ. , and a CCD electronic camera for acquiring the image of the antibiogram, said apparatus preferably being equipped with a calculation unit for determining on said image the inhibition circles around each patch and thus calculating the concentration minimum inhibitory effect for each antibiotic tablet, characterized in that it is equipped, for a given culture medium and a given germ, with at least one optical filter adapted so that the camera can acquire the image of the antibiogram in a determined wavelength range, said range being determined so as to maximize the Q ratio defined by the following formula:

Q = Rs (λ - Rμ (λ) Rs (λ) where Rs (λ) is the reflection coefficient of the germ-free culture medium, as a function of the wavelength λ and Rg (λ) is the reflection coefficient of the same medium, but seeded with the germ, as a function of the wavelength λ. For a culture medium of the Miller Hinton type, the wavelength range can be between approximately 0.5 and 0.6 μm, and preferably less than 0.55 μm. Alternatively, the range can be in the near infrared, preferably between 1 and 1.3 μm.

According to another characteristic of the invention, the abovementioned range is determined so as to maximize both the abovementioned Q ratio and the Q 'ratio defined by the following formula:

Q '= Ts (λ) - Tg (λ ^ Ts (λ)

where Ts (λ) is the transmission coefficient of the germ-free culture medium as a function of the wavelength λ and Tg (λ) is the transmission coefficient of the same medium, but seeded with the germ, as a function of the length wave λ. Advantageously, the filter or filters are interference filters interposed on the field of observation of the camera.

In a particular embodiment, the camera is a matrix or linear CCD electronic camera. In this case, the lighting system may include lighting means for focusing light rays towards the surface of the box, the incident rays forming an oblique angle with respect to the surface to be illuminated of the box, so that the reflected rays pass in the vicinity of the camera lens, but do not penetrate it.

To better understand the object of the invention, we will now describe, by way of purely illustrative and nonlimiting examples, an embodiment shown in the accompanying drawing. On this drawing :

- Figure 1 is a partial schematic view in vertical elevation of a particular embodiment of the apparatus of the invention; FIG. 2 is a graph representing the response curve of a low-pass interference filter, as a function of the transmission coefficient of the light intensity and the wavelength;

- Figure 3A is a graph showing the response curves of a transparent culture medium with and without germ, as a function of the reflection coefficient of the light intensity and the wave number;

- Figures 3B and 3C are graphs similar to Figure 3A, but respectively for a culture medium supplemented with blood and for a culture medium supplemented with a pigmentation enzyme;

FIG. 4A is a graph representing the response curves of a transparent culture medium with and without germ, as a function of the transmission coefficient of the light intensity and the wave number; and

- Figure 4B is a graph similar to Figure 4A, but for a culture medium supplemented with a pigmentation enzyme.

In FIG. 1, the apparatus of the invention comprises a linear CCD electronic camera 1, the observation plane 2 of which is oblique with respect to the surface of a Petri dish B. A filter 3 is inserted on the plane d observation 2 of the camera 1. A lighting means consisting of a rectilinear bar 4 sends a light brush 5 towards a portion of the box B, the mean line of the light brush 5 being oblique with respect to the surface of the box . The rectilinear strip 4 can be adapted to receive a bundle of optical fibers, the downstream ends of which are distributed throughout the strip, so as to define a line of light points which sends a dihedral-shaped lighting brush over the portion of surface to be lit from the box. Translational drive means are provided for moving the dish B in the direction of the double arrow F, so that the camera 1 can acquire the image of the entire surface of the agar contained in the dish B.

As a variant, the camera 1 could be replaced by a matrix CCD electronic camera whose observation axis coincides with the axis of the box B, and the rectilinear bar 4 could be replaced by a ring circumscribed in the box B, in axial projection, to illuminate the entire surface of the box. In this case, it is not necessary to move the box B, to acquire the image of the entire surface.

Figures 3 and 4 show the results of spectrometric measurements in reflection and transmission carried out using a Fourier transform spectrometer, on culture media with and without germ.

For each dish containing a given culture medium, two measurements were carried out: a first measurement relates to a sample of the culture medium without germ; a second measurement relates to a sample of the culture medium where a germ has grown. The first measurement corresponds substantially to a measurement which would have been carried out on an inhibition zone around an antibiotic tablet.

FIG. 3A shows the results of two tests carried out on a transparent culture medium of the Muller Hinton type. For the first test, the curve Rs1 corresponds to the response curve in reflection of the culture medium without germ, and the curve Rgl corresponds to the response curve in reflection on the same medium with a germ. We note that the curve Rsl has a peak whose coefficient of reflection R amounts to approximately 16%, for a wave number N of between approximately 18,500 and 19,000 cm ^"1. The curve Rgl also has a peak for the same wave number, but the reflection coefficient R of this peak is around 3%. To optimize the contrast in reflection between the zones with germ and the zones without germ of the culture medium, it is not so much the difference between the two curves that matters, but the ratio between the difference of the two curves and one of the two curves. For the second test, we also plotted the curve Rs2 for the response of the germ-free culture medium and the curve Rg2 for the response of the culture medium with germ The peaks of these last two curves correspond substantially to the same wave number as the peaks of the first two curves.

We also note that these curves have another peak in the vicinity of a wave number of the order of 9,000 cm ⁻¹ , which corresponds to the near infrared. In FIG. 3B, three tests were carried out on a medium of transparent culture of the Muller Hinton type supplemented with blood from sheep, which gives a red color to the agar. In the same way as for the tests illustrated in FIG. 3A, the curves in FIG. 3B all have a peak for a wave number between 18,500 and 19,000 cm ^"1 , as well as another peak in the vicinity of 9,000 cm ^"1 . On the other hand, the reflection coefficient R for the peaks is lower, because the agar is red in color.

In FIG. 3C, a single test was carried out on a transparent culture medium of the Muller Hinton type pigmented with a germ of the pseudomonas aeruginosa type, which gives a green color to the agar. In this case, the reflection curve Rg of the culture medium with germ has a peak rising to around 6% in reflection coefficient for a wave number between 18,500 and 19,000 cm ^-1 . The response curve Rs of the germ-free culture medium has a practically zero reflection coefficient, with a slight peak of the order of 0.3% for the same wave number. The curve Rd represents the difference between the curve Rg and the curve Rs. Another peak is also present for a wave number comprised between approximately 9,000 and 9,500 cm ⁻¹ .

It can be seen in FIG. 3C that the reflection coefficient is lower for a green agar than for a transparent or red agar. Although the results are not illustrated, the reflection coefficient is still lower for a "chocolate" agar, that is to say a transparent agar supplemented with heated blood.

The wavelength N is the inverse of the wavelength λ. Thus, the range of wave numbers between 18,500 and 19,000 cm ^-1 corresponds to a range of wavelengths between about 526 and 540 nm, and the range of wave numbers between 9,000 and 9,500 cm ^"1 corresponds to the wavelength range between approximately 1.050 and 1.11 nm. Thus, for the peak located between 18,500 and 19,000 cm ^"1 , an interference filter can be used, the response curve of which is illustrated. in FIG. 2. This filter has a transmission coefficient T greater than 90% for a wavelength λ less than about 550 nm. In FIG. 4A, the curve Ts represents the transmission response of a transparent culture medium without germ, while the Tg curve represents the response of the same culture medium with germ. It can be seen on this graph that the difference between the curves Ts and Tg increases for a decreasing wave number, between 30,000 and 18,000 cm ^"1. It is important to also take into account the transmission coefficient of the culture medium, because when l 'a black background is placed under the plate containing the agar, the light is absorbed in the zones of inhibition of the culture medium, to improve the contrast with the zones where the germ has grown.

Thus, for a wave number between 18,500 and 19,000 cm ⁻¹ , the difference in the transmission coefficient is acceptable.

In FIG. 4B, the same test was carried out for a transparent culture medium pigmented with a germ of the pseudomonas aeruginosa type, which gives a green color to the agar. The curve Td represents the difference between the curve Ts and the curve Tg. It is also found that, for a range of wave numbers between 18,500 and 19,000 cm ⁻¹ , the response in transmission is acceptable.

However, in practice, it is necessary to find a compromise between the best response in reflection and the best response in transmission of the culture medium. Although the invention has been described in conjunction with several particular variant embodiments, it is obvious that it is in no way limited thereto and that it includes all the technical equivalents of the means described as well as their combinations, if these these are within the scope of the invention.

Claims

1. Apparatus for automatically reading an antibiogram, said apparatus comprising a lighting system (4) for lighting a Petri dish (B) containing antibiotic pellets positioned on a culture medium seeded with a germ, and a CCD electronic camera (1) for acquiring the image of the antibiogram, characterized in that it is equipped, for a given culture medium and a given germ, with at least one optical filter (3) adapted so that the camera (1) can acquire the image of the antibiogram in a determined wavelength range, said range being determined so as to maximize the ratio Q defined by the following formula:

Q = Rsfλ) - Rg (λ ⁾ Rs (λ)

where Rs (λ) is the reflection coefficient of the germ-free culture medium, as a function of the wavelength λ and Rg (λ) is the reflection coefficient of the same medium, but seeded with the germ, as a function of the wavelength λ.

2. Apparatus according to claim 1, characterized in that the wavelength range is between about 0.5 and 0.6 microns, and preferably less than 0.55 microns.

3. Apparatus according to claim 1, characterized in that the range is included in the near infrared, preferably between 1 and 1, 3 microns.

4. Apparatus according to one of claims 1 to 3, characterized in that the aforementioned range is determined so as to maximize both the aforementioned Q ratio and the Q 'ratio defined by the following formula:

Q '= Ts (λ - Tg (λ)

Ts (λ)

where Ts (λ) is the transmission coefficient of the germ-free culture medium as a function of the wavelength λ and Tg (λ) is the transmission coefficient of the same medium, but seeded with the germ, as a function of the length wave λ.

5. Apparatus according to one of claims 1 to 4, characterized in that the filter or filters (3) are interference filters interposed on the field of observation (2) of the camera (1).

6. Apparatus according to one of claims 1 to 5, characterized in that the camera (1) is a matrix or linear CCD electronic camera.

7. Apparatus according to claim 6, characterized in that the lighting system comprises lighting means (4) for focusing light rays (5) towards the surface of the box (B), the incident rays forming an angle oblique to the surface to be illuminated of the box, so that the reflected rays pass in the vicinity of the camera lens (1), without however penetrating therein.

8. Apparatus according to one of claims 1 to 7, characterized in that it is equipped with a calculation unit for determining on said image the inhibition circles around each patch and thus calculating the minimum inhibitory concentration of each antibiotic tablet.