Friday, August 31, 2007

Aging in Bacteria Redux

Earlier I posted on aging in Bacteria. Today I received an email from Moselio Schaechter, author of the most excellent blog, Small Things Considered. I reprint the email's contents here (with his permission of course).

"About your exciting posting on aging in bacteria, I am reminded of an experiment that I did perhaps 50 years ago. I have no record of it and can only remember the results in general terms.

In an early day preoccupation with the meaning of "balanced growth," it became crucial to have faith in the homogeneity of Salmonella cultures, which speaks to the question bacterial senescence. The experiment could not have been simpler, and had probably been done by others many times. I coated a glass slide with agar, put a thin suspension of cells on it, waited for the liquid to be imbibed, checked under a high dry objective (with phase contrast) to see that the cells looked reasonably single, incubated until ca. 5-6 generations had passed (in rich medium, ca. 2 hours), and scored the number of cell that had not made a microcolony. The answer, best I can remember, is that there were none in the fields examined. I don't remember how many microcolonies I had scored, but it must have been at least 1000. So, under the conditions and by this criterion, there were less than 0.1% non viable cells. Incidentally, my recollection is that the microcolonies were quite uniform in the number of cell each. I walked away thinking that there may not be much senescence in my bugs.

As data go, this is useless at this point, not having anything on paper (and perhaps undergoing senescence myself). However, the experiment can be easily and cheaply repeated using robotic colony counters that can measure the number of cells/microcolony. The results can be obtained in a couple of hours. The level of sensitivity can be quite high, perhaps 1 in 10exp5 or better..

Let's say this is true. It suggests that perhaps the difference between Stewart's result and this one is agar. I have no problem imagining a mechanical obstacle to old cell poles, something that may not matter in liquid medium. Also, cell crowding may be a factor, although it doesn’t show up in his work. As for Caulobacter, even in liquid, I can't really fathom if this is relevant because of the different life styles of the two bugs."

The interesting thing is that the work I have been doing for the past year requires immobilising E. coli on glass slides with poly-lysine and taking movies of them over time. My object was to observe phage lysis times, but conceivably the process could be modified to conduct the work Schaechter suggests. In fact, I might already have the data, albeit encoded in movies waiting to be deciphered.

So what to make of Schaechter's comments. Unfortunately without Schaechter's data, it is difficult to make comparisons between the two studies. However, Stewart et al. write:

"During the growth of the microcolonies, sixteen cells were observed to cease growing; these cells never resumed growth during the course of the experiment. We have defined these cells as potentially dead cells and have analyzed their locations in the lineages. While these apparent deaths may ultimately be due to stochastic events, they show a statistically significant bias (p = 0.01; see Materials and Methods) toward increased divisions spent as an old pole (over the total observation history)."

Given that Stewart et al. observed 35,049 cells, the fraction that "died" is 4.6exp-4.... far below Schaechter's estimated 0.1%. Perhaps we should be asking why did so many cells in Schaechter's experiment die? (Update 9/1/07: From Schaechter: Sorry, but, best I remember, I didn’t see any die. I just guessed that their upper limit had to be less that 1/1000.) Schaechter's impression that microcolony were quite uniform in number of cells suggests that these microcolonies will contain both "old" and "young" cells, and that their growth rates are similar. The question is, given an initial population that supposedly contains both old and young cells, are the differences in growth rate between cells sufficient to produce large differences in microcolony sizes when microcolonies are initiated on agar? This sounds like a great question for Eric Stewart. Presumably he has this data since he tracked microcolonies by film.

Figure above from Stewart et al. 2005. Legend below.
Figure 2. Average Lineage Showing Old Pole Effect on Growth RateThe first division in the microcolonies is not represented, as the identity of the poles is not known until after one division (hence each initial cell gives rise to two lineages that are tracked separately, and subsequently combined from all films to create the single average lineage shown here). The lengths of the lines connecting cells to their progeny are proportional to the average growth rate of that cell; a longer line represents a higher growth rate for that cell. At each division, the cell inheriting the old pole is placed on the right side of the division pair, and shown in red, while new poles are placed on the left side of each pair, and shown in blue (note that this choice of orientation is not the same as that of Figure 1, to compare more easily old and new pole lineages). Because the position of the start of the growth line for each new generation is dependent on the generations that preceded it, the difference in growth rates is cumulative. Green lines indicate the point at which the first cell divides in the last four generations. Nine generations from 94 films encompassing 35,049 cells are included in this tree. The average growth rate of all the cells corresponds to a doubling time of 28.2+/−0.1 min. The data used to generate the average lineage are provided in Dataset S1.

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