Classic Spotlight: What's on (in) Your Plate Today?

Atypical day for many microbiologists begins with a trip to the incubator to check the growth of their cultures. The careful researcher will record the nuances of growth such as the size, shape, color, and number of colonies on solid media or the turbidity of broth cultures. Depending on the growth rate of the organisms in question, this process may be repeated several times a day. The data produced by these growth experiments speak volumes about the biology of the cells under culture. Indeed, it is remarkable what we can learn from such simple data. Consider, for example, how the observation of diauxie during growth of Escherichia coli on media containing both glucose and lactose led to the discovery of transcriptional gene regulation and the operon hypothesis (1) or how the purification of compounds that stimulated growth allowed the discovery, quantification, and functional characterization of many vitamins, including coenzyme A (2), lipoic acid (3), coenzyme M (4), and vitamin B12 (5). Sadly, however, many of us never consider the basic composition of the media we use, although I suspect we would all agree that this profoundly impacts the results of our experiments. Admittedly, we often think about which antibiotic to include or whether rich or minimal medium would be more appropriate, but be honest: have you ever considered what nitrogen, phosphorus, and sulfur sources are present in your medium? How about the trace element composition, the choice of buffer, or the concentration of salts? My guess is that you have not or that, if you did, you fell into the trap of “more is better than less,” despite the fact that high concentrations of nutrients are almost never observed in nature or that many are toxic at elevated levels. Fortunately, there are those among us who have not fallen into the trap, as exemplified by a classic paper by Neidhardt and coworkers published in the Journal of Bacteriology (JB) in 1974 (6) that describes the development of one of the first truly defined culture media. In this respect, I should be clear: numerous prior publications report media that are fully “defined” with respect to their molecular components. What sets the JB paper apart is the careful analysis of the levels of each component needed to support maximal growth rates with reasonable growth yields. Thus, the researchers established growth-limiting levels not only of carbon but also of nitrogen, phosphorus, sulfur, magnesium, and iron. Numerous other medium components were also examined, and while growth limitation for most micronutrients was not observed, the experiments clearly established the levels at which toxic effects were manifest. The researchers emphasized the importance of the specific strains chosen for their study, which they recognized as being responsible for much of the variation observed between different laboratories working with the same species. Also significant was their careful attention to the establishment of conventions for handling strains, which allowed highly reproducible growth experiments. The result of this painstaking effort was a culture medium for enteric bacteria that can accurately and reproducibly distinguish between growth rates that differ by as little as 1.4 min per generation (6). Moreover, the use of a truly defined medium permits facile substitution of the carbon, nitrogen, sulfur, and phosphorus sources and allows labeling experiments using isotopes of these elements (7). This feature was immediately exploited by the authors, revealing the surprising findings that E. coli can use sulfonic acids as a source of sulfur and that the addition of low levels of bicarbonate eliminates the pronounced growth lag that occurs when small numbers of cells are inoculated into fresh medium. Intriguingly, neither trait is observed with the close relative Salmonella enterica. Since its introduction, the MOPS (morpholinepropanesulfonic acid)-buffered medium has been used in thousands of experiments, leading to a detailed understanding of the metabolism of numerous carbon sources and the kinds of phosphorus, nitrogen, and sulfur molecules that support growth of both S. enterica and E. coli (8). Further, this medium enables highly accurate growth predictions using modern metabolic modeling methods (9), a feature that was presciently envisioned in the original publication (6). There is little doubt that our thorough understanding of these model organisms is based in large part on the types of experiments enabled by this truly defined medium. The profound impact and ongoing legacy of this basic enabling research can be seen in the fact that this classic manuscript continues to be cited at least once per week, now more than 40 years after its original publication! So, I ask: what’s in your plate today?