STUDIES IN THE PHYSICO-CHEMICAL BEHAVIOR

OF BACTERIA

Allen E. Stearn and Esther Wagner Stearn

CHAPTER I

INTRODUCTION

The authors have, in a series of researches carried on during the past few years, (references 105 to 111 inclusive) studied various aspects of the physico-chemical behavior of bacteria. In these studies interest in ultimate chemical constitution was confined to its bearing on the physico-chemical nature of the organism.

It is the purpose of the present study to bring together, from a unified point of view, the conclusions reached in these various studies, to amplify somewhat the experimental foundations of these previous conclusions, and to present certain experimental results bearing on aspects of the subject which have not hitherto been emphasized.

The experimental results obtained up to the present have furnished a rational basis for general theories regarding staining behavior, including specific effects of individual decolorizers and the function of so-called mordants, bacteriostatic behavior toward anions and cations, and electrophoretic behavior.

The general point of view adopted in interpreting the data has been that we are dealing with systems which, when any environmental condition is altered, tend to a new equilibrium condition. The term equilibrium is not used here in the sense of maximum thermodynamic stability entirely. Any equilibrium condition obtaining in a bacterial system, whether the organism be dead or alive and especially in the latter case, will be a highly labile and dynamic state, and will represent only a condition of metastability. The term is used then partly in the sense of basal plane. Expressed more definitely and in chemical terms, the word equilibrium is used to represent that type of equilibrium expressed by the mass law.

Whatever may be said of the general validity of this law, it has been found to furnish a means, at least in the case of the so-called weak electrolytes, of quantitative predictions which are in accord with experimental data obtained by methods of fair precision.

It is not proposed to discuss the soundness of this law, but it should be pointed out that there are some who claim, on the basis of good and of fairly extensive experimental evidence, that even in the cases where most people think that the mass law does not yield even approximately correct results, this is due, not to an improper formulation of the law, but to a lack of knowledge as to the actual magnitudes to be substituted into the equations. Thus, while there is defined a hypothetical thermodynamic magnitude called the "activity" for the purpose of "making the mass law hold", Smith and his co-workers, in a series of researches, have shown that a proper knowledge of the actual concentrations of the particular molecular species involved

in the equation under consideration renders the use of the activity function unnecessary (though such findings do not militate against its usefulness as a thermodynamic function),102 103

The components of the bacterial cell which seem important in determining its physico-chemical nature and behavior are such as may be considered, if they undergo chemical reactions of the ordinary ionic type at all, to "obey" the mass law with a precision well within that of ordinary biological measurements.

It may, perhaps, be argued that, in considering living organisms, one should confine oneself, outside of empirical determinations of actual chemical compositions, to a study of the chemical processes which are uniquely vital, i. e. reactions characteristic of the organism in its "live" state and confined to such a state. From such a point of view a dead organism is a much too complicated system to study as a whole, and the benefit to be derived from a chemical study of the organism is largely confined to the elucidation of the transformations it brings about in the substances which compose its environment. In the case of a rather simple and comparatively undifferentiated organism it is almost necessary, then, that one tacitly assume that the function of prime chemical interest on the part of such an organism is the secretion of enzymes which "direct" all the real work, and that the organism is not itself otherwise a part of the reacting system. Even such apparently specific and selective reactions as those catalyzed by enzymes (speaking now from the point of view of the organism and its natural environment only) may be found to be more general than might at first be supposed. Thus Vaughan states128 that bacteria secrete enzymes, as do all living cells, for the purpose of disrupting, for example, the protein molecule constituting its food. Those enzymes "will digest certain proteins but not all proteins. If a living cell is in contact with a foreign protein against which it does not possess a digestive ferment it will gradually evolve a ferment specific for that protein". He states his belief that it is a fundamental law that "a living cell in contact with a foreign protein will evolve an enzyme to destroy that protein".

Thus metabolism, as he continues, "is regulated by environment. Reaction between the organism and its environment is essential to all living matter." It is with these immediate reactions between the organism and its environment, or more specifically with the shifting of the metastable equilibria of the organism in response to regulated changes in environment, that the present study deals.

In a certain very restricted sense an organism may be pictured almost as a single molecule in its responses. On this point Vaughan128 also makes the statement that "whether the individual organism consists of a single molecule or many molecules I do not know... I know of no way of distinguishing between intermolecular and intramolecular activity".

A second type of question as to the importance of work on the behavior of such complicated systems as bacteria, other than a study of the chemistry of their metabolism, should be briefly considered. If our interest is centered in the chemical system as such without much regard to whether it be "alive" or "dead", might it not be far better to attempt to synthesize systems simulating the structure and composition of the bacterial systems as far as possible but with compositions completely known? Might not conclusions finally reached from a study of such systems be less speculative than those reached after studying the bacterial systems as nature

provides them? In answer to such a possible question two points should be noted. In the first place, as will be shown experimentally in Chapter II, the validity of the picture which will be formulated more fully later but which is vaguely suggested here for the postulated bacterial system, tends to be confirmed, not only qualitatively but also quantitatively, by the behavior of simple synthesized systems, as far as the analogy can be carried experimentally with the systems studied. Even though this is the case, one would not be warranted in applying results obtained from a study of very simple chemical systems to the much more complicated bacterial systems. It is rationally necessary that one refrain from postulating a mechanism of purely chemical behavior which is contradicted by a study of simple, comparatively completely known systems, but the justification for picturing the more complicated system as structurally analogous to the simpler one exists only in case it is experimentally found to react to a variation of the same factors in an analogous manner. From this point of view much valuable information may be obtained by a study of the bacterial cell as such a system. The type of equilibrium in which we are interested is not "frozen" by the fact of an organism's being in the "live" condition. As has been so aptly stated 127, "when matter becomes endowed with life it does not cease to be matter; it is not released from the laws which govern its structure, its attractions and its motions. In studying living things it should be borne in mind that they are material in composition and subject to the fundamental laws that govern matter, and possessed of those properties essential to matter."

CHAPTER II

GENERAL THEORY

A. OUTLINE OF POINT OF VIEW

In a preliminary paper10% the authors reported that a culture of Bacterium coli, inoculated into lactose broth containing basic dye, precipitated out, upon incubation, yielding a precipitate only very faintly colored, and that the medium during the incubation increased in acidity. The same culture, inoculated into nutrient broth, yielded, upon incubation, a dark colored precipitate of bacterial matter, leaving the medium, which in this case was eventually decolorized, more alkaline. On the other hand, when acid dyes were used in the fermenting lactose media, the bacterial precipitate resulting from the growth of the Bacterium coli was more intensely colored than when these acid dyes were used in plain nutrient broth. The changes in the reaction of the two types of media, lactose broth and plain nutrient broth, were, however, the same as when basic dyes were employed.

This simple experiment indicated possible combination of the bacterial substance with basic dye which increased in extent as the alkalinity of the medium increased, and also combination with acid dye increasing as the acidity was increased. There was thus indicated a pH at which bacteria might not combine with either dye, a pH which would possess the characteristics of an isoelectric point for the bacterium as a whole, and these results were explained on the basis of this idea.

Further work soon revealed the fact that a pH, characteristic of a particular bacterial strain, and possessing properties similar to those of an isoelectric point, could be easily found, but that the behavior of the organism was such that this point differed somewhat from the isoelectric point of a single amphoteric chemical individual.107. At the same time it was recognized that the bacterial system was too complicated to be pictured as a single chemical individual without inquiring into the probable behavior of simpler systems of more than one component.

A second approximation in the picture was made, and much subsequent work has not altered this second picture appreciably.

The bacterial system behaves, as will be shown in the following chapters, as an equilibrated system of two classes of ampholytes, or, in the simplest possible case, of two ampholytes one of which is distinctly more acidic than the other. If we have two such ampholytes, the more acidic with an isoelectric point I, and the other with a corresponding point I', then, when this system is in a solution whose hydrogen ion concentration is greater than I, both components will act as bases and combine with anions. When the system is in a solution whose hydrogen ion concentration is less than I', both components will act as acids and combine with cations. When the hydrogen ion concentration is between I and I' the one component will act as a base and the other as an acid, and they will tend to combine with each other. The isoelectric point of the system as a whole will lie somewhere between the two values I and I', which range may be called the isoelectric range. For simple systems this resultant isoelectric point can be calculated if we know the acidic and basic strengths of the two components and their relative amounts.