Classic Spotlight: Plate Counting You Can Count On

EDITORIAL

In every introductory microbiology class in the United States (and, I expect, the world) at some point early in the course the professor or teaching assistant introduces the next generation of microbiologists to the concept of using CFU to determine the viable bacteria in a culture. Instructors explain the concept of serial dilution, as well as the process of finding that dilution which provides a readily countable number of colonies on a plate. Most textbooks and lab manuals typically recommend using plates that yield somewhere between 30 and 300 colonies (or some similar range). Too few colonies, and the count may not be accurate; too many colonies, and it is difficult as well as time-consuming to distinguish the individual colonies on a plate. The plating method for counting viable bacteria is considered general knowledge, and as such, when asked why we use this method it is easy to respond, “Well, this is just the way that it's done.”

It turns out that counting colonies was once a somewhat unsettled topic and was addressed in published works as far back as 1895 (1). Breed and Dotterrer sought to resolve this issue in a paper published in the Journal of Bacteriology in 1916 (2), the first year of the journal's publication. The first sentence of the introduction comes to the central point of the paper quickly by stating, “A point which is of much importance in making bacteriological counts is the limit in the number of colonies that may be allowed to grow on a plate without introducing serious errors.” The authors then note, “Probably every bacteriological worker has this point in mind in making counts and has his own opinion based on experience; but there are few published data on the subject.” Resolving this point was a serious matter, as these counts were used in analyzing milk for bacterial contamination. Furthermore, it was known at the time that the colony sizes of the various microbes in milk varied a great deal (with some organisms yielding pinpoint colonies) and that some organisms could enhance the growth of neighboring colonies. In their report, Breed and Dotterrer conclude that “the group of plates having more than 40 and less than 200 colonies per plate” provided the most reproducible findings with the least variability. In contrast, the authors state that “it is at once clear that plates having less than 20 and more than 400 colonies are so apt to be widely discrepant that counts from plates of this sort should be disregarded.” Their study, which required 1,435 agar plates (these were the reusable glass petri plates!), forms the foundation of the general knowledge we still teach today.

The views expressed in this Editorial do not necessarily reflect the views of the journal or of ASM.