Imaging Bacterial colonies

Counting bacterial colonies is a great means of identifying the general bacteria content of a sample. Growing bacteria cultures on a gelled-growth medium, like an agar plate or petri dish, permits researchers identify colonial characteristics. Bacteria or other microorganism colonies can be observed by scientists through various staining and imaging methods. The process of imaging bacterial colonies is made simple by using gel imagers, such as the Azure Imaging Systems.

A simple workflow for imaging bacterial colonies includes:

  1. Pipetting the bacterial sample on an agar plate
  2. Spreading the sample evenly
  3. Incubate: wait for colonies to grow on the surface
  4. Stain colonies with crystal violet
  5. Capture images using a capable imager or scanner
  6. Count and analyze your colonies using analysis software
How to image bacterial colonies on an agar plate
Imaging bacterial colonies begins with pipetting bacterial sample onto an agar plate, and ends with analyzing the results from an imaging system using AzureSpot Pro, or another analysis software. (Created with

Capturing fluorophores that fluoresce in the visible spectrum is useful many applications. Here are some examples of when capturing fluorophores might be useful: 

  • On-plate screening for gene insertions or mutations,
  • Protein-protein interaction studies,
  • Gene transfer studies,
  • Characterization of colony morphology, or 
  • Verification of fluorescent protein expression.

Why is crystal violet staining used for cell counting?

Crystal violet is used to measure cell viability of adherent cells. Using crystal violet is an easy and efficient method for the quantification of cell density in cell culture plates. 

Does crystal violet stain live or dead cells?

During the crystal violet assay, dead cells are washed away and the remaining attached live cells are visualized with the crystal violet dye. This assay method works by the utilization of protein-DNA binding that results in the staining of the nucleus dark blue and the cytoplasm light blue. Crystal violet absorbs at 595 nm.

Benefits of using crystal violet staining

The crystal violet assay process occurs quickly, providing fast results and reliable readings. With this assay, it is possible to determine the number of cells in a culture, as well as their concentration, characteristics, and behavior. It is a highly recommended technique for researchers and scientists who depend on the analysis of cell culture density in their work.

By using crystal violet assay, researchers can save time and resources, while also obtaining highly accurate data that can further their understanding of various biological processes and phenomena.

Using GFP in colony counting

GFP stands for green fluorescent protein. GFP is a protein produced by the bioluminescent jellyfish Aequorea victoria (also known as “crystal jelly”). As its name suggests, GFP emits bioluminescence in the green zone of the visible spectrum. In modern day, GFP is cloned and commonly used in molecular biology as fluorescent markers of gene expression. It emits bright green light (lambda max = 510 nm) when excited with ultraviolet (UV). GFP is expressed in many species, including yeasts, bacterium, fish, fungi, and plants. A dish of GFP-expressing marijuana is shown in Figure 1.

GFP-expressing marijuana
Figure 1. GFP-expressing Marijuana imaged with an Azure imager

Main advantage of GFP in cellular biology

An advantage of GFP expression is that it is highly sensitive and thus can be used to visualize primary cellular functions, such as signal transduction, protein translation, and DNA replication.

What machines can I use to image bacterial colonies?

Imaging bacterial colonies can be done using laser scanners, like the new Azure Sapphire FL Biomolecular Imager, or Western blot imaging systems, like the Azure Imaging Systems. The Sapphire FL enables imaging and quantitation of several multi-well plates at a time with a 25x25cm scanning area, ensuring the absorbance of each well easily measured. Azure Imagers can also be used to image more than just gels and membranes.

Scientist looking at bacterial plate before placing in Azure Sapphire
The Azure Sapphire Biomolecular Imager has a large field of view (25x25cm) that enables imaging and quantitation of several multi-well plates at a time. Scan as many as 6 membranes (9 x 7 cm) or 6 96-well plates at a time.

Because of the depth of field, the Azure Imagers can capture images of bacterial or yeast colonies on plates. Every Azure Imager can capture white light images, but only the Azure 400 and 600 Systems can image fluorophores that fluoresce in the visible spectrum. These two imagers allow you to capture of microorganisms expressing fluorophores, such as GFP (Figure 1), mCherry (Figure 2), or both (Figure 3).

An agar plate with E. coli expressing GFPmut3 (green) and mCherry (red). The plate was imaged using red and green LEDs in the Azure 600.
Figure 2. An agar plate with E. coli expressing GFPmut3 (green) and mCherry (red). The plate was imaged using red and green LEDs using the Azure 600 Imager.
GFP- and mCherry-expressing E. coli.
Figure 3. Bacterial plate of GFP- and mCherry-expressing E. coli. imaged using an Azure Imager

Other sources of fluorescence on a flat surface can be captured by imagers, including bacterial colonies on a plate. Fluorescent proteins, like GFP variants, fall in the visible range. Digital imagers, like the Azure 400, can be used for more than just gels and membranes.  The Azure 600 is a great for image bacterial colonies on plates because it offers two-channel NIR fluorescent detection.

What software is used to count bacterial colonies?

AzureSpot Pro colony counter bacterial plate imaging, data analysis software
AzureSpot Pro features a variety of analysis modules give users powerful analysis tools at their disposal, including modules for: Multiplex and single-channel gels and blots, plates and arrays, area analysis, profile analysis, and colony counting.

There are multiple analysis software options available on the market to count bacterial colonies. AzureSpot Pro is an in-depth analysis software that includes a Colony Counter module, making the process of counting of spots or features fast and easy. Download a free trial of AzureSpot Pro by clicking here.

Systems used to image bacterial colonies
AzureSpot Pro colony counter bacterial plate imaging, data analysis software

Analysis software for the analysis of plates, gels, cell-based assays, and more

All-around application ace: NIR, RGB, chemi, blue light, white light, and UV

Related Applications

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