There are some techniques in the life sciences laboratory that have stubbornly remained as tedious, multistep, manual tasks, despite the increasing use of automation in the lab. Colony counting as part of a clonogenic assay is one such technique. In this article, we summarise what cell culture colony counting can tell us, and how the manual technique is usually conducted, followed by the reasons for automating this method, and how we might do so.
What is colony counting in a clonogenic assay?
In tissue culture, one of the most important pieces of information that the clonogenic assay can give us is an idea of the reproductive health of a cell. To form a colony, a cell needs to survive any treatment that we have given them, and then successfully divide into viable daughter cells in the medium we choose to grow them in. As a simple example, cells might be treated with a drug, and after some time, the fraction of cells that remain viable are determined by counting the colonies that have formed. A more complex example might be to generate two cell lines, one with a mutation in the ABC gene, and then to determine whether the mutation enables the cells to overcome inhibition of a specific biochemical pathway to survive and reproduce.
Traditionally, cells are seeded onto 10 cm tissue culture plates at a very low density (to ensure that individual colonies can be distinguished), and then incubated until surviving cells have produced colonies of more than 50 cells. A colony with over 50 cells is generally visible to the naked eye, particularly after staining the live cells with a dye like methylene blue. A digital colony counter is sometimes used to help remember which colonies have been counted and what the running total number is, by beeping and incrementing a digital counter when a scientist identifies a colony with a press of a marker pen. Because so few cells can be seeded on each plate, large numbers of plates are needed to ensure that results are reliable, and this makes manual colony counting both time consuming and error prone.
What is the impact of errors in colony counting?
Counting colonies manually means that the data generated can be subjective, and human error can occur if a scientist is distracted, tired, or in a rush. Unlike machines, humans are susceptible to limitations in terms of attention, perception, memory, and logical reasoning1. Errors such as slips, lapses, and mistakes can happen to even the most experienced and well-trained scientist2.