In the course of some studies on the kinetics of growth and the biochemical activities of a marine diatom it became desirable to obtain bacteria-free cultures. The classical method of obtaining such pure algal cultures involves either repeatedly washing single cells in sterile medium or obtaining discrete bacteria-free algal colonies by growth on a solid medium. Both these methods have been widely applied to fresh-water species by Pringsheim (1946) and others, whilst Chu (1946) has used both methods with the marine diatom Nitzschia closterium (Ehrenberg) Wm. Smith forma minutissima.

Little is known of the biochemistry of diatoms, although many workers have reported growth experiments with the unicellular algae of the marine phytoplankton. Experiments have often been performed without due regard for the appropriate control of physical and chemical conditions. Many reports contain only incomplete data of the growth under a given set of conditions, and it is often impossible to say whether the effects recorded are upon the growth rate, the total crop, or both. Other studies have been reported which included the addition of organic matter to cultures which were only uni-algal and not bacteria-free. At the present time even the mere maintenance of stock cultures of the marine unicellular algae is perforce an empirical matter. Results in replicate cultures often show gross differences in growth that are apparent on inspection by eye alone, and insufficient information is available regarding the nature of these variations in growth to allow the rational development of improved culture media. Due therefore to a lack of suitable techniques, most of the results available are difficult, if not impossible, to interpret in terms of the biochemical activities of the algae.Quantitative measurements of the growth of bacterial cultures have been widely used by Hinshelwood, Monod and others (Hinshelwood, 1946;Monod, 1942). The technique may be limited in its application, for example in the elucidation of metabolic pathways by the permeability of the cell wall to intermediates, but such studies can furnish much valuable information. Investigations have shown that the growth of bacteria as measured by an increase in cell numbers or cell material generally obeys certain simple rules. The growth can thus be defined by a number of growth constants which reflect the physiological behaviour of the cells. Similar considerations should apply to the growth of any unicellular organism. Comparison of the growth constants under various conditions should therefore provide some information about the suitability of these conditions for culture work, allow the rational development of improved culture media, and make possible fuller interpretation of the results of growth experiments.

(Text-figs. 1-10) 127 In recent years, ethylenediamine tetra-acetic acid (EDT A) has been extensively used in culture media for unicellular algae. This, and other chelating agents are also becoming widely used in toxicity studies with plants and animals, in analysis and for controlling the ionic concentration of certain metal ions in a wide variety of studies.EDT A forms complexes with a large number of metallic cations, and in the presence of sufficient EDT A only very small amounts of many metals can exist as free ions. The use of EDT A as a metal buffer system has been advocated by numerous workers. These metal buffer systems depend for their operation upon the equilibrium between the metal cations, the EDT A and the chelate. Providing that the concentrations of the chelate and the che1ating ion are large compared with that of the free metal ion, such a system will tend to maintain a constant concentration of metallic ions, should these be continuously removed by a biological system.The available data suggest that EDT A is not readily metabolized by most forms of life, and moreover it does not appear to be markedly toxic except by virtue of its meta1-che1ating reactions. Its use under the correct conditions for controlling metal ion concentrations in biological systems therefore has much to commend it.The physical chemistry of metal-EDT A equilibria has been examined by several workers (Chaberek, Bersworth & Martell, 1955;Raaflaub, 1956) and methods for calculating the concentrations of free metal ion at equilibrium outlined. These treatments, however, are limited to simple cases when only one chelating metal ion is present. The biologist is inevitably faced with working with systems containing many reacting metal ions (e.g. in sea water). Data regarding the free metal ion concentrations existing in equilibrium with EDT A in such systems is of obvious importance if the use of EDT A is to be fully exploited. This paper records some data on the equilibria set up when EDT A is added to sea water and on the effect of variations of certain controlling factors.