THE TUBE METHOD OF MEASURING THE GROWTH RATE OF NEUROSPORA

IN CONNECTION with investigations on the biochemical genetics of the ascomycete Neurospora, a technique has been developed for studying growth by measuring the progression of mycelial frontiers within horizontal glass tubes half filled with suitable solid culture media. This method is the equivalent of measuring the increase in radius of a circular colony, a method used by many workers in studying growth of fungi, bacteria, and tissue cultures. The tube method is particularly useful in studying growth rates of rapidly growing organisms such as Neurospora for which petri plate cultures of unwieldy dimensions would be required. Horizontal glass tubes for limiting mycelia to growth in one direction were used by Fawcett (1925) in determining growth rates of Pythiacystis citrophthora and Diplodia natalensis. It has been pointed out how "pyridoxinless" and "aminobenzoicless" mutant strains respond in such growth tubes to varying amounts of the vitamins pyridoxin and p-aminobenzoic acid, which these strains are unable to synthesize (Beadle and Tatum, 1941). While it is clear that this method is not universally applicable in bioassay procedures and not entirely free of disadvantages, it is nevertheless a method by which, under certain conditions, growth responses can be measured with precision. Accordingly, it is important to know in some detail the effects of various factors such as hydrogen ion concentration, temperature, etc., on this particular type of growth response. It is the primary purpose of this paper to supply such information. In planning the experiments reported, the use of the method for bioassays has been kept in mind, and attention has been directed particularly to such factors as might be expected to affect the method in this application. A "growth tube" assay of yeast extract for p-aminobenzoic acid has been made to indicate in principle how mutant strains might be used in bioassay work. THE ORGANISM AND ITS NUTRITIONAL REQUIREMENTS. -Under the name Monilia sitophila the imperfect stage of Neurospora has long been known as a mold infesting laboratories and bakeries. Several species are recognized (Shear and Dodge, 1927). N. sitophila and N. crassa are heterothallic, producing sexual spores only if the two mating types are present, while N. tetrasperma is heterocaryotic and facultatively heterothallic. These three species have been used in genetic studies following the demonstration by Shear and Dodge (1927) that their life cycles are particularly well adapted to such studies. One important advantage of Neurospora shared by 1 Received for publication July 17, 1943. Work supported by grants from the Rockefeller Foundation and from the Penrose Fund of the American Philosophical Society. 2 National Research Council Fellow, now at Columbia University, Department of Zoology. other fungi and many other organisms is that strains are readily propagated asexually and consequently can be maintained genetically constant as long as contaminations or mutations do not occur. Of the three species, crassa has been most extensively studied genetically, particularly by Lindegren (see review, 1942). The species sitophila, crassa and tetrasperma are able to grow on synthetic media containing inorganic salts, an inorganic nitrogen source, a suitable source of organic carbon, and the vitamin biotin (Butler, Robbins and Dodge, 1941, Beadle and Tatum, 1941). With the exception of biotin, all vitamins, amino acids, and other factors required are synthesized by the organism. Because Neurospora can be grown on a chemically defined medium, it has been possible to establish and detect mutant strains unable to synthesize particular vitamins, amino acids, and other substances. Such mutants, of which three have been described, are obtained as a result of X-ray or ultraviolet treatment. They are detected in a manner described elsewhere (Beadle and Tatum, 1941). With few exceptions, each of the mutant strains so far analyzed requires that only one specific substance be added to the so-called minimal medium. After obtaining the "pyridoxinless" and "thiazoleless" mutants in N. sitophila, a change to N. crassa was made because of its higher sexual fertility and because more is known about its genetics. The studies included in the present report, accordingly, are to a large extent confined to wild-type strains of N. crassa. In earlier experiments a single ascospore strain obtained from Dr. C. C. Lindegren was used. This strain is of mating type A (+ in the terminology of Lindegren). It is designated as "regular" and was used unless otherwise indicated. In later experiments a recently isolated single ascospore strain derived from "regular" was used and designated Number 1. This strain, also mating type A, has the same or nearly the same inherent growth rate as the "regular" one. Strain R977 grows somewhat faster and is of mating type a (-). Other strains are described in connection with the particular experiments in which they were used. Occasional reference to N. sitophila is made. In both form and rate of growth, the two species are so similar, however, that it is almost certain that the conclusions resulting from the strains on crassa will apply equally to sitophila. THE TUBE TECHNIQUE IN GENERAL. -The growth tubes adopted as a standard in the work reported here are made of 40 cm. lengths of pyrex glass tubing with an internal bore of about 13 mm. Terminal segments 5 cm. in length are bent up at an angle of 450, and the openings are fire polished and flared slightly (fig. 1). Such tubes can be held upright on