Application Note

Automated colorimetric red-white colony screening on the QPix 400 System

  • Increase efficiency by automating red-white colony screening
  • Rapidly and accurately screen and pick red-white colonies
  • Enhance color contrast between red-white colonies

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Introduction

Colorimetric screening is a simple but effective method for selecting recombinant bacteria. A common colorimetric detection method, blue-white colony screening, utilizes the interaction between ß-Galactosidase and X-gal to distinguish which bacteria contains the gene of interest. Our QPix™ 400 System has improved laboratory efficiency by automating blue-white screening using the QPix Chroma Colorimetric Colony Selection software module and the red QPix Chroma Filter for bluewhite colony selection. The system’s colorimetric screening capabilities have been expanded to perform red-white colony screening using the QPix Chroma Colorimetric Colony Selection software module in combination with the new blue QPix Chroma Filter (Figure 1). This is useful when working with naturally red organisms or red-colored alternatives to X-gal. In this application note, we demonstrate how to perform red-white colony screening with the QPix 400 system.

Figure 1. The new blue QPix Chroma Filter used to enhance contrast between red and white colonies.

Preparing the red and white colonies

E. coli, the most commonly used bacterial model organism, was used as an example of a white organism, and Serratia marcescens D1, which expresses red pigmentation when grown at room temperature, was used as an example of a red organism.

Both organisms were grown aerobically in a shaker for 24 hours. E.coli was cultured in Luria broth at 37°C, while S.marcescans D1 was cultured in tryptic soy broth at 25°C. After incubation, the E.coli and S.marcescans D1 cultures were mixed together in tryptic soy broth. This mixture was spread onto a tryptic soy agar (TSA) Nunc™ OmniTray™ and incubated for 2 days at 25°C.

Imaging the red and white colonies

To capture an image of the red and white colonies, the blue QPix Chroma Filter was placed on the imaging light table of a QPix 420 and a QPix Microplate Holder was placed over the filter. Then, the TSA OmniTray was inserted into the QPix Microplate Holder.

“Blue-White Picking” from the QPix Chroma Colorimetric Colony Selection module was used to distinguish between red and white colonies (Figure 2). Normally, this module is used to screen between blue and white colonies, but by using the blue QPix Chroma Filter instead of the red QPix Chroma Filter, the QPix software can be adapted to screen for red and white colonies (Figure 3A). The blue QPix Chroma Filter enhanced the color contrast between the E.coli and S.marcescens D1 colonies which allowed for easier discrimination of the two types of colonies. (Figure 3B).

Figure 2. The main menu for the QPix 420 software. The Blue-White Picking process (circled in red) is located under Picking Processes in the QPix 420 software.

Figure 3. A TSA OmniTray containing E. coli colonies (white spots) and S. marcescens D1 (red spots). An image of the TSA OmniTray without the blue QPix Chroma Filter (A) and with the blue QPix Chroma Filter (B) is shown.

Red and white colonies were automatically identified and selected using the built-in “Auto Select” feature on the QPix 400 software (Figure 4). The “Blue Auto Select” setting was used to identify the red S. marcescens D1 colonies (Figure 5C), while the “White Auto Select” setting was used to identify the white E.coli colonies (Figure 5D). When viewing the standard color camera photo (Figure 5A), the QPix 400 software properly identified the red and white colonies. The software was even able to properly identify the light pink S. marcescens D1 colonies. Using the blue QPix Chroma Filter further enhanced the color contrast (Figure 5B). After identification of the red and white colonies, the QPix 400 accurately and rapidly picked the selected colonies.

Figure 4. QPix 400 software Blue-White Auto Select feature. After capturing a Test Image, the Auto Select feature can be used to automatically identify red (by selecting blue) or white colonies. The histogram threshold for determining red or white colonies can also be adjusted manually. These detection settings will be used to identify red or white colonies for the entire sample.

Figure 5. Zoomed in image of the TSA OmniTray from Figure 2. A) A standard color camera photo showing the white colonies (E. Coli) and colonies showing various shades of red (S. Marcescens D1). B) A QPix white light image using the blue QPix Chroma Filter. C) The QPix 400 software red colony detection and (D) white colony detection applied on the QPix white light image. The yellow masks indicate colonies that met the picking selection criteria.

Conclusion

The QPix 400 series offers a sophisticated solution for colorimetric colony screening through a simple user interface and fast, precise robotics. Using the blue QPix Chroma Filter, the QPix Chroma Colorimetric Colony Selection software module can be adapted to include red-white colony screening.

ProductConfigurationCatalog #

QPix 420/450/460High-throughput screening and objective selection system for microbial coloniesContact Us

QPix Chroma Colorimetric Colony Selection Software LicenseBlue/White Colony Selection Software License for QPix 400 SeriesSL9400-A06

2 Way Adjustable Microplate HolderPlate compatibility: any ANSI/SLAS 1-2004 microplatesX9402

Blue QPix Chroma Filter. Thin film optical filter for Red/White colony selection1 filterContact Us

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