More Effective Colony Counting and Area Measurement

Problems in colony counting and area measurement

Microbial and genotoxicity testing are implemented to ensure product safety. In microbial testing, the number of colonies of live bacteria in a medium is counted at specified time intervals and the number of bacteria is calculated for evaluation. In one type of genotoxicity testing, the Ames test, mutation induction is evaluated according to the colonies and their proportions formed when a test substance is given to a strain that originally cannot produce amino acids on its own.
In both tests, it is necessary to distinguish and measure the colonies of live cells and bacteria that proliferate and change with time on a well plate. However, with visual colony counting by testers, it is virtually impossible to analyze and evaluate all well plates without variation or errors in values. Another significant problem is that it is difficult to immediately understand the status of an entire well plate during observation and counting of colonies under high magnification over a small field of view. This time-consuming work also presents a risk of simply missing parts of a well plate.

In regenerative medicine research, the observation of entire well plates and accurate analysis of colony formation of iPS cells are essential for quantitative evaluation of experiment results. To make the cultivation process better and more efficient, it is also important to accumulate data in each step from cultivation to medium exchange and passage under different conditions.
In colony counting, variations in results from operator to operator are often a factor preventing accurate data accumulation. This problem is more likely to occur when iPS cells proliferate in the maintenance and cultivation processes, because the shape of the cells in the colonies change. The boundaries between the cells may blur, which makes visual counting more difficult, causing human error during measurement and evaluation.

This problem has further reaching impacts: when a mistake in manual counting or false detection in automatic counting is found, it takes a lot of time and effort to perform analysis and testing again. To prevent the wasting of time and effort of a limited workforce for inspections, tests, and experiments, and produce as many results as possible, both data accuracy and work efficiency must be improved.

Solutions for colony counting and area measurement

KEYENCE’s All-in-One Fluorescence Microscope BZ-X800 has the versatility to perform fluorescence, brightfield, and phase contrast observations and various imaging methods with a single unit. The BZ-X800 can observe entire well plates, perform quantitative colony counting and area measurement, and solve various problems in experiments, research, tests, and inspections.
This section introduces the advantages of using the BZ-X800 and various application examples.

Example of streamlining iPS cell colony counting and area measurement over an entire well plate

Live cells and bacteria change over time, which affects the results of well plate analysis and evaluation, so it is important to obtain quantitative analysis results quickly.
The following images, captured with the BZ-X800, show an example of automatic colony counting and area measurement using Hybrid Cell Count on the image of an entire well plate of iPS cells obtained using image stitching. The number of colonies, average area, standard area deviation, and total area can be measured quantitatively from the wide-field-of-view observation of an entire well plate, which provides analysis results quickly. This can save a huge amount of the time and effort required for manual counting and also eliminates the risk of human error.

1. 
3. 

   Quantity

   251

   Average area

   8,315 µm²

   Standard area deviation

   12,652 µm²

   Total area

   2,086,967 µm²

Objective lens: CFI60 CFI Plan Apo λ 10x
Image stitching: 7 images x 9 images

When a multi-well plate is used, the BZ-X800’s Macro Cell Count automatically processes a batch of multiple images under the conditions extracted for a single image.
Conventional colony analysis over multiple wells requires a huge amount of time and effort, and presents the risk that analysis results and evaluation will vary from operator to operator. With the BZ-X800, quantitative measurement and analysis can be achieved easily and quickly.

Batch measurement under the same conditions