Taking a total microbial count or total viable count is a regular procedure in clinical, food, dairy, and pharmaceutical microbiology. Traditional manual methods for enumerating bacterial colonies (or colony forming units) are labor-intensive and tiresome processes requiring a high concentration level. Colony counter provides an alternative sustainable solution to this problem by helping in fast and accurate counting of colonies.
A colony counter is an instrument used to count colonies of bacteria or other microorganisms growing on an agar plate. Various types of colony counters are available to help enumerate colonies of bacteria and yeast quickly and accurately. Some of these colony counters are manual, while others are automatic.
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Manual Colony Counters
Manual counters/semi-automatic counters depend on the ability of a technician to see colonies distinctly and mark them with a special type of pen on the outer surface of the plate. The machine keeps the count of such marked colonies. Though this method helps to some extent, it is still time-consuming, hectic, and prone to errors.
Principle of Manual colony counter
A model of a manual colony counter works by placing a Petri plate on an electronic pressure pad with light illumination and marking each colony by touching the plate with a felt tip pen. The pressure of the touch registers a count in the digital display. The pressure is adjustable according to the requirement. This model helps in avoiding the mistake of missing or double-counting colonies. Wolfhuegal graticule, segmentation disc, and centering adapters for 50-90 mm plates are also provided in the instrument. There are some added features like the facility of dark background for translucent colonies, optimum viewing of colonies by peripheral, glare-free illumination, and a built-in averaging facility to count multiple plates in this model. The software generates the average colony count, and using a toggle switch background is switched. The data/counted colony is transferred to the computer using USB connectivity.
Colonies may be crowded and small, which makes counting difficult. So dividing the counting into small squares and magnifying the colonies with the help of magnifying glass mounted on the flexible arm makes the work somewhat easier. This model for counting colonies allows significantly low output and is time-consuming. Also, when more than one technician counts, the total count of colonies may vary.
A bacterial colony is a group or cluster of bacteria derived from one common bacteria.
Automatic Colony Counters
The automatic bacterial colony counter uses image processing algorithms such as gray scaling, thresholding, filtering, etc., to count colonies efficiently.
Principle of Automatic Colony Counters
A fully automatic colony counter collects images using a digital image capturing devices such as a document scanner, digital camera, webcam, charge-coupled device (CCD), or video equipment. Counting colonies automatically starts with capturing an image of the Petri plate with colonies. It is followed by digitizing the image using software packages. Finally, the last step is processing the digitized image using a single/multi-threshold segmentation procedure to separate and detect the colonies. Automation of the colony counting method will help preserve time. Those laboratories that do not have the colony counter can send the images via the internet to the laboratory having the analyzing software.
The contrast of the objects and the transparency of their background vary widely in an automated system. One of the following methods of illumination is selected to enhance the visibility and accuracy in counting colonies;
- Transmission method: For routine high contrast objects with relatively transparent backgrounds.
- Dark-field method: For high contrast objects with an opaque background.
- Reflection: For low contrast objects and fairly transparent background.
The modern instrument with a 1.4 megapixel CCD can capture HD (high definition) color images. Likewise, a typical automated colony counter consists of red, green, and blue long-life LEDs, which help receive good color images with brilliant contrast without chromatic aberrations. The black, white, and transparent background is available for effective bright field and dark field exposure for the sample platform. Both round-plate of 55-150 mm and square-plate of 150 mm×150 mm can be used. It can measure colonies as minute as 43 μm or zones accurately to 0.1 mm.
Petri dishes free of reflection and shadow is difficult to illuminate because they are transparent and reflective. Multiple lighting could create artifacts on the edge of the agar, which are likely to be counted as a colony. It leads to inaccuracy in counting, affecting the instrument’s precision. Interscience for microbiology has developed a white diffusing dome for 360°lighting without reflections and shadows o overcome the problem. Their Scan® 4000 model incorporates this feature.
Advantages of Automated Colony Counter
Automated colony counter has many advantages. Some of them are:
- It takes less than 10 seconds to take a picture of a Petri plate with colonies, and the software analyzes it quickly. Hence decreasing the time required for counting.
- Since software counts colonies, it increases accuracy and reduces manual labor.
- The counting of multiple plates becomes hassle-free.
References
- Khandpur, R. (2019). Colony Counter, Automated. Compendium Of Biomedical Instrumentation, 491-494. https://doi.org/10.1002/9781119288190.ch95
Colony counter