WO2001022080A2

WO2001022080A2 - System and method for distinguishing microbial colonies/plaques

Info

Publication number: WO2001022080A2
Application number: PCT/US2000/025702
Authority: WO
Inventors: Jeffrey P. Woessner
Original assignee: Paradigm Genetics, Inc.
Priority date: 1999-09-20
Filing date: 2000-09-19
Publication date: 2001-03-29
Also published as: WO2001022080A3; AU7983900A

Abstract

System and device that uses a colored filter for readily distinguishing between blue and white microorganism colonies/plaques that are subjected to blue-white screening is disclosed.

Description

SYSTEM AND METHOD FOR DISTINGUISHING MICROBIAL COLONIES/PLAQUES

Field of the Invention The present invention relates to a highly effective system and method for enabling reliable and accurate visual and/or automated differentiation between blue and white microbial colonies/plaques.

Background of the Invention Experiments involving biotechnology include the use of biological systems such as genetically engineered microorganisms. In order to prepare such microorganisms, new genetic material usually in the form of DNA

(deoxyribonucleic acid) fragments, is inserted into the microorganism. If successfully inserted, the genetic makeup of the microorganism will be changed so that it can express a new trait (i.e. making a new protein). However, merely providing the new genetic material to the microorganism does not guarantee that the material has actually been inserted into or incorporated by the microorganism. A classical biochemical test, known as blue-white screening, has been used to differentiate between microorganisms that have incorporated the new genetic material and those that have not. In the blue-white screening test, microorganism colonies that have incorporated the new genetic material should appear white, whereas microorganisms that have not incorporated the genetic material should appear blue. Although the blue and white colonies are visible to the human eye, such colonies or plaques are sometimes difficult to distinguish under laboratory conditions where the blue color is not fully developed. Further, even when the blue color is fully developed, automated devices that include, inter alia, cameras equipped with colony differentiating software, are frequently unable to accurately differentiate between blue and white colonies (often hundreds to thousands on a plate, pinhead sized or smaller). It would be highly desirable to provide a system for easily distinguishing between blue and white colonies/plaques by visual inspection and/or automated equipment. Thus, a new approach was sought to readily distinguish or differentiate among microorganism colonies/plaques that are subjected to blue-white screening.

Summary of the Invention In one embodiment, the present invention is directed toward a system for readily distinguishing between blue and white microorganism colonies/plaques that are subjected to blue-white screening, comprising: a) microorganism colonies/plaques that are subjected to blue-white screening, b) a light source, c) means for detecting said microorganisms/plaques, and d) at least one color filter positioned i) between said microorganism colonies/plaques and said light source, and/or ii) between said microorganism colonies/plaques and said detection means, wherein said color filter improves the contrast between said blue and white microorganism colonies for detection by said detection means.

In another embodiment, the present invention is directed toward a method for readily distinguishing between blue and white microorganism colonies that are subjected to blue-white screening, comprising: a) using microorganism colonies/plaques that have been subjected to blue- white screening, b) providing a light to said microorganism colonies/plaques for detection by detection means, c) providing at least one color filter positioned i) between said microorganism colonies/plaques and said light source, and/or ii) between said microorganism colonies/plaques and said detection means, wherein said color filter improves the contrast between said blue microorganism colonies and said white microorganisms; and d) detecting said blue and white microorganisms with said detection means. Preferably, the blue and white microorganisms are in a culture medium. Preferably the culture medium is a culture dish with a growth medium. Also preferred is that the source of color is a red filter. Preferably the red filter is contiguous to said culture medium or said culture dish. Also preferred is that the red filter is on a camera lens used to inspect or scan for said blue and white microorganism colonies. Preferably, the means for detecting said colonies/plaques is a camera having a camera lens and software for distinguishing among the blue microorganism colonies/plaques and the white microorganism colonies/plaques. Also, the detection means can also be visual inspection by the aided or unaided human eye.

In another embodiment, the present invention is directed toward a system for readily distinguishing between blue and white microorganism colonies/plaques that have been subjected to blue-white screening, comprising: a) microorganism colonies/plaques that are subjected to blue-white screening; b) a light source; c) means for detecting said microorganisms/plaques; and d) light filter means positioned i) between said microorganism colonies/plaques and said detection means and/or ii) between said microorganism colonies/plaques and said light source, wherein said light filter means shifts the grey scale used by the detection means to differentiate among said blue and white microorganism colonies/plaques.

In another embodiment, the present invention is directed towards a method for readily distinguishing between blue and white microorganism colonies/plaques that have been subjected to blue-white screening, comprising: a) using microorganism colonies/plaques that are subjected to blue-white screening, b) providing a light to said microorganism colonies/plaques for detection by detection means, c) providing at least one light filter positioned i) between said microorganism colonies/plaques and said detection means and/or ii) between said microorganism colonies/plaques and said light source, wherein said light filter shifts the grey scale used by the detection means to differentiate among said blue and white microorganism colonies/plaques; and d) detecting said blue and white microorganism colonies/plaques with said detection means.

DETAILED DESCRIPTION OF INVENTION Colored filters useful with the present invention include red, orange, yellow and green filters. Preferably, the colored filter is a red filter. Suitable red filters include 23A Light Red filter, 25 Red 1 filter and 29 Dark Red filter. Such filters can be obtained from any commercial source of photographic equipment. Similarly, filtration can also be achieved using colored acetate sheets, such as those used to package floral arrangements and displays. The net effect of such filters is to alter the color of the light entering the detector, thus effectively shifting the grey scale, i.e. the spectrum of black and white images that the detector actually "sees" on the plates. White and blue colonies/plaques are normally seen close together on a grey scale. A light or colored filter shifts the images for blue and white colonies/plaques such that they are farther apart on the grey scale. Such shifting apart or broadening of the black and white images on the grey scale enables detection means or software packages to better or more accurately differentiate among the colonies/plaques.

The term "colony" refers to a contiguous source of single cells, such as bacteria, derived from a single ancestor and growing on a solid surface. The term "plaques" refers to clear, round areas on an otherwise opaque layer of bacteria or tissue-cultured cells where the cell have been infected with a virus. Such plaques tend to form slight depressed areas on growth media. The term "colonies/plaques" refers to microorganism colonies and/or plaques collectively.

The term "light source" refers to any light source that can transmit sufficient light to one side of a plate or substrate containing microorganism colonies or plaques to allow reading, detection, differentiation or distinguishing of microbial colonies or plaques on the other or opposite side of the plate or substrate. Preferably, the light source is substantially white light. However, a colored light source may conveniently be employed which may serve as both the light source and filter in one device. For example, a red light bulb could be employed that would provide a source of red light via filtration by the bulb glass. The term "detection means" refers to any suitable means for detecting the colonies and plaques. Although the aided or unaided eye may be used for such detection, preferably such detection is made by an automated reader or camera lens coupled to a software package for detecting the colonies or plaques.

Fig. 1 represents a system for distinguishing blue-white colonies/plaques using a color filter located over the camera lens. Fig. 2 represents a system for distinguishing blue-white colonies/plaques using a color filter located between a culture plate and a light source.

In Fig. 1 , system 1 is made up of light-emitting source 4, support 18, culture plate 6, microbial colonies/plaques 8, camera 10, color lens 12, detecting means 14 (including computer and software) for interpreting the data input from camera 10. Microbial colonies/plaques 8 (colonies shown as exaggerated mounds and plaques as depressions, not shown) that contain a treatment for blue-white screening are grown on culture media 6a within culture plate 6. When colonies/plaques 8 are sufficiently developed for detection, culture plate 6 is placed upon support shelf 18 having a transparent medium (i.e. clear glass) or is resting in a mask placed on the shelf. Light from light-emitting source 4 passes through the following: transparent glass 18, culture plate 6, culture media 6a, microbial colonies/plaques 8, color filter 12, camera lens 10a and finally into camera body 10. The colored filter, preferably red, darkens blue objects and the white ones are left white. This filtering effect yields a dramatic improvement in the ability of detecting means 14 to distinguish or discriminate between white and blue colonies/plaques in culture plate 6. In Fig. 2, system 2 is made up of light-emitting source 4, support shelf 18, color filter 12a, culture plate 6, microbial colonies/plaques 8, camera 10, detecting means 14 (including computer and software) for interpreting the data input from detecting means 14. Microbial colonies/plaques 8 that contain a treatment for blue-white screening are grown on culture medium 6a within culture plate 6. Color filter 12a lays flat on support shelf 18. Thus, at least a portion of color filter 12a is beneath culture plate 6 and culture medium 6a. Alternatively, support shelf 18 can use a colored medium that serves as the filter. As a third option, culture plate 6 can be made of a colored material (i.e. a red plastic or glass) which can also serve as a filter. When colonies/plaques 8 are sufficiently developed, culture plate 6 is placed upon support shelf 18. Light from light- emitting source 4 passes through the following: transparent medium or aperture 18a, colored filter 12a, culture plate 6, culture media 6a, microbial colonies/plaques 8, camera lens 10a and finally into camera body 10. The colored filter, preferably red, darkens blue objects and the white ones are left white. This filtering effect yields a dramatic improvement in the ability of detecting means 14 and software 16 to distinguish or discriminate between white and blue colonies/plaques in culture plate 6.

Example 1 . Distinguishing blue-white colonies with a red filter located between microbial colony and light source

A. Preparation of blue-white microorganism colony. Vectors with a polycloning site within the coding region of a part of a β-galactosidase gene (lac Z) allow blue-white screening. A peptide comprised of the 146 N-terminal amino acids encoded by this truncated lac Z gene can associate with the carboxy-terminal region of β-galactosidase. Insertion of a DNA fragment into the polycloning site disrupts the production of a functional N-terminal peptide, preventing complementation to form an active β-galactosidase. Active β-galactosidase can cleave the X-gal molecule to yield a blue color. Vectors containing an insert (i.e. a fragment of DNA) can thus be detected by the loss of β- galactosidase activity as indicated by the lack of a blue color in cells containing the vector. The preparation of a representative blue-white colony screening is as follows. Four hundred (400) microliters of 2% X-gal (5- bromo-4-chloro-3-indolyl-β-D-galactopyranoside) in dimethyl formamide and 150 microliters of 20% IPTG (isopropylthio-β-D-thiogalactopyranoside) made up in water are added to 200 milliliters of molten LB agar for each 22 x 22 cm square plate. Stock solutions of X-gal (20 milligrams/milliliter) in dimethyl formamide are wrapped in aluminum foil and stored at -20 degrees Celsius (°C). IPTG stock solutions (2 g IPTG in 8 ml of water) are stored frozen at - 20 °C. The bacteria transformed with the vector are spread onto the agar plate with a glass rod and allowed to dry onto the surface. The plate is inverted and incubated at 37 °C for 12-16 hours. Colonies containing vector with no insert should appear as blue (i.e. active β-galactosidase) and those with an insert should appear a milky white. White colonies may be picked manually with a toothpick or robotically using pins, inoculated into growth media and tested by plasmid DNA isolation, restriction digest to release the

DNA insert and agarose gel electrophoresis. Picked colonies/plaques may also be screened by polymerase chain reaction (PCR) using primers flanking the polycloning site. Distinguishing the blue and white microbial colonies/plaques on an automated robotics platform. One or more sheets of red-colored acetate, such as those found used for floral wrapping, are placed over the light source or the support shelf of the Qpix colony picker (trademark of Genetix, Dorset, United Kingdom). By placing individual sheets down on the light deck of the Qpix and resting the agar plate containing the blue and white microbial colonies to be imaged on top of the red colored acetate sheets, excellent contrast between the blue and white microbial colonies/plaques is obtained. The blue color in the colonies or plaques is darkened or enhanced and the white colonies are left white. This filtering effect yields a dramatic improvement in the ability of the automated imaging software to discriminate between white and blue colonies/plaques. Subsequently, the automated robot is able to more accurately pick only the desired white colonies/plaques. C. Distinguishing between blue and white colonies using visual inspection. By placing individual sheets down a light box and resting an agar plate containing the blue and white microbial colonies/plaques to be manually picked on top of the red colored acetate sheets, excellent contrast between the blue and white microbial colonies/plaques is obtained. The blue color in the colonies or plaques is darkened or enhanced and the white colonies are left white. This filtering effect yields a dramatic improvement in the ability of the human eye to visually discriminate between white and blue colonies/plaques. This insures accurate picking of only the desired white colonies/plaques.

Example 2. Distinguishing blue-white colonies with a red filter located between microbial colony and detection means

The same conditions are practiced as in Example 1 , except that sheets of red colored acetate are omitted and a Light Red 23A filter is attached to the camera lens that is part of the detection means. The filtering effect yields a dramatic improvement in the ability of the automated imaging software to discriminate between white and blue colonies/plaques.

Claims

WHAT IS CLAIMED IS:

1. A system for readily distinguishing between blue and white microorganism colonies/plaques that have been subjected to blue-white screening, comprising: a) microorganism colonies/plaques that are subjected to blue-white screening; b) a light source; c) means for detecting said microorganisms/plaques; and d) at least one color filter positioned i) between said microorganism colonies/plaques and said detection means and/or ii) between said microorganism colonies/plaques and said light source, wherein said color filter improves the contrast between said blue microorganism colonies/plaques and said white microorganism colonies/plaques for detection by said detection means.

2. The system of claim 1 wherein b) said light source emits substantially white light.

3. The system of claim 1 wherein c) said means for detecting said microorganism colonies/plaques comprises a camera having a camera lens and software for distinguishing among said blue microorganism colonies/plaques and said white microorganism colonies/plaques.

4. The system of claim 1 wherein said color filter is positioned I) between said microorganism colonies/plaques and said detection means.

5. The system of claim 3 wherein said color filter is attached over said camera lens of said camera.

6. The system of claim 3 wherein a red color filter is attached over said camera lens of said camera.

7. The system of claim 3 wherein a 23A Light Red filter is attached over said camera lens of said camera.

8. The system of claim 1 wherein said color filter is positioned ii) between said microorganism colonies/plaques and said light source.

9. The system of claim 8 wherein a red color filter is employed.

10. A method for readily distinguishing between blue and white microorganism colonies/plaques that have been subjected to blue-white screening, comprising: a) using microorganism colonies/plaques that are subjected to blue-white screening, b) providing a light to said microorganism colonies/plaques for detection by detection means, c) providing at least one color filter positioned i) between said microorganism colonies/plaques and said detection means and/or ii) between said microorganism colonies/plaques and said light source, wherein said color filter improves the contrast between said blue microorganism colonies/plaques and said white microorganism colonies/plaques; and d) detecting said blue microorganism colonies/plaques and said white microorganism colonies/plaques with said detection means.

1 1. The method of claim 10 wherein b) said light source emits substantially white light.

12. The method of claim 10 wherein d) said means for detecting said microorganism colonies/plaques comprises a camera having a camera lens and software for distinguishing among said blue and white microorganism colonies/plaques.

13. The method of claim 10 wherein d) said detecting means is a visual inspection.

14. The method of claim 10 wherein said color filter is positioned i) between said microorganism colonies/plaques and said detection means.

15. The method of claim 12 wherein said color filter is attached over said camera lens of said camera.

16. The method of claim 12 wherein a red color filter is attached over said camera lens of said camera.

17. The method of claim 12 wherein a 23A Light Red filter is attached over said camera lens of said camera.

18. The method of claim 10 wherein said color filter is positioned ii) between said microorganism colonies/plaques and said light source.

19. The method of claim 18 wherein a red color filter is employed.

20. The method of claim 19 wherein said red color filter is located under said microorganism colonies/plaques.

21. A system for readily distinguishing between blue and white microorganism colonies/plaques that have been subjected to blue-white screening, comprising: a) microorganism colonies/plaques that are subjected to blue-white screening; b) a light source; c) means for detecting said microorganisms/plaques; and d) light filter means positioned i) between said microorganism colonies/plaques and said detection means and/or ii) between said microorganism colonies/plaques and said light source, wherein said light filter means shifts the grey scale used by the detection means to differentiate among said blue and white microorganism colonies/plaques.

22. A method for readily distinguishing between blue and white microorganism colonies/plaques that have been subjected to blue-white screening, comprising: a) using microorganism colonies/plaques that are subjected to blue-white screening, b) providing a light to said microorganism colonies/plaques for detection by detection means, c) providing at least one light filter positioned i) between said microorganism colonies/plaques and said detection means and/or ii) between said microorganism colonies/plaques and said light source, wherein said light filter shifts the grey scale used by the detection means to differentiate among said blue and white microorganism colonies/plaques; and d) detecting said blue and white microorganism colonies/plaques with said detection means.