INTRODUCTION

THE HISTORICAL PROBLEM is how to quantitate the vigor of a given culture so that it can be compared to cultures growing under other conditions. Our two methods presented here are based on a culture's expenditure of energy as indirectly indicated by the size of its ATP pool. While direct quantitation of cellular ATP is time consuming and expensive, one of our two methods is both fast and inexpensive for Gram-negative bacteria.

The idea began with a need to compare cultures that had been giving others unintelligible results. Attardi and others thought that the closer the lac-operon was to oriC in E. coli translocation mutants, the more ß-galactosidase should be expressed based on gene dosage in bi-directionally replicating genomes. However, the data obtained was a random collection of points.

In the early 1980's, one of us (CWV) suggested the prevailing notion of "log-phase" was in error, and gave rise to the aforementioned spurious data. For several years, he had given his undergraduate students the exercise of plotting growth curves using both abs_550nm (biomass) versus time, and cfu/ml versus time - something all microbiology students worth a grade should be able to do. Without fail the lines were never even closely parallel with cfu/ml rising faster than biomass - the cells were becoming smaller! - meaning that the average cell was in fact rapidly varying over the commonly-held "log-phase."

We wish now to report how combining those conditions with two quantitative methods for indirectly assaying ATP pools produces a means for assessing the relative "health" of cells in a culture.

With more dilute cultures, it was found that they would suffer increasing stress due to O₂ depletion once they penetrated a threshold of 2x10⁷ cfu/ml, above which O₂ was used faster than it could dissolve. When transposition mutants were assayed under these rather dilute conditions, a line having a negative slope resulted in a plot of lactase/cell versus distance from oriC (Fig. 1).
Figure 1: Direct correspondence between lac-expression and gene dosage in steady state cultures of E.coli lac-transposition mutants (1).

TECHNOLOGICAL REASONING

In 2003, lac-operon expression levels were tested versus the osmotic stressor sucrose, and were shown to be inverse to [sucrose], presumably due to adenyl cyclasešs production of cAMP, which is inverse to [ATP] as ATP was partially being diverted to osmoregulation (2).

A linearity exists between growth rate versus cellular specific gravity existed (3) because synthesis of dense ribosomes depends on pGppp-sensitive transcription of rrn genes, and [pGppp] is indirectly related to the concentration of activated amino acids, which is, in turn, proportional to [ATP]. Thus cells with higher ribosome content (range: 15-45% dry wt of cell) are physically denser (spec. grav.: 1.32 - 1.42).* (Gram-negative bacteria are, by the way, totally permeable by sodium halides.)

Below are presented comparisons of the methods outlined in the last two points above. Both were found to be fast, and the last particularly inexpensive and of better precision.

* Because the term "density" can be confused within bacteriological circles for cell population density, the authors will hereafter use the similar and less confusing term "specific gravity."

With more dilute cultures, it was found that they would suffer increasing stress due to O₂ depletion once they penetrated a threshold of 2x10⁷ cfu/ml, above which O₂ was used faster than it could dissolve. When transposition mutants were assayed under these rather dilute conditions, a line having a negative slope resulted in a plot of lactase/cell versus distance from oriC (Fig. 1).
	Figure 1: Direct correspondence between lac-expression and gene dosage in steady state cultures of E.coli lac-transposition mutants (1).