William J. Robbins

Cultivation of Excised Root Tips and Stem Tips Under Sterile Conditions

1. A simple method of growing the isolated meristematic tissue of higher plants, excised root tips and stem tips, under sterile conditions is described. 2. The excised root tips of peas, corn, and cotton make considerable growth in the dark in solution cultures containing mineral salts and glucose or levulose. 3. The excised root tips of peas, corn, and cotton make little growth in the dark in solution cultures containing mineral salts and lacking carbohydrate. 4. The growth of the isolated root tips of peas, corn, and cotton is markedly greater in solution cultures containing glucose than in those containing levulose. 5. The excised roots of corn respond normally to gravity when grown on agar containing mineral salts and glucose. 6. The isolated shoot tips of peas and corn make considerable growth in the dark in sterile solution cultures containing mineral salts and glucose or levulose, but little in the absence of carbohydrates. 7. The excised shoot tips of corn and peas grown in sugar solutions remain chlorotic, and those of peas show the stem elongation and small leaf development characteristic of plants grown in the dark. 8. When the excised root tips of corn are grown for ten days or two weeks in the dark in a solution culture containing glucose and mineral salts, and the tip is then cut off and transferred to a fresh solution of the same type, the amount of growth in the second period is less than that in the first, and ceases in the third period.

Studies on Euglena and Vitamin B 12

Hutner et al (1949) reported that Euglena gracilis var. bacillaris exhibits a quantitative growth response to crystalline antipernicious anemia factor (vitamin B12) This discovery makes it possible to use Euglena as a means of bioassay for this factor. The following presents some results on vitamin B12 as determined from the growth of Euglena gracilis. Materials. The culture of Euglena gracilis var. bacillaris employed was kindly supplied by Dr. S. H. Hutner. Three media were used. The composition of Medium I and Medium II we owe to the courtesy of Dr. Hutner and Dr. T. H. Jukes. Medium I (acid) and II (neutral) were used for the cultivation of Euglena. Medium III was devised for the detection of organisms which synthesize B12. Medium I, in addition to mineral salts, contained thiamine, glutamic acid and malic acid. Its pH was adjusted to 3.0. It was prepared by mixing suitable amounts of three stock solutions.

Effect of Light on Growth of Excised Root Tips Under Sterile Conditions

1. Diffuse light was favorable to the continued growth of root tips in a modified Pfeffers solution containing 2 per cent glucose, or the same solution to which 80 ppm or 400 ppm of autolized yeast were added. 2. Anthocyan developed in some of the excised corn roots in the light, but in none in the dark. 3. The anthocyan pigment was present in the root cap, at points where secondary roots were to emerge or had emerged, and at the base of the root, but not in the meristematic region. 4. The longest period a root tip was grown was 149 days, through ten periods of transfer, in diffuse light in the modified Pfeffers solution containing 2 per cent glucose and 400 ppm of autolized yeast. 5. Roots which remained attached to the grain during the first two weeks grew for a longer time than those grown under excised conditions from the start. One such root was grown in the dark for 133 days, 119 in excised condition.

Effect of Potato Extracts on Growth of Phycomyces

1. Evidence has been presented which confirms previous reports that spore germination, early growth, and gametic reproduction in Phycomyces may be stimulated in cultures to which a potato extract has been added. Increasing amounts of potato extract in the culture medium up to a certain amount may increase growth and reproduction; quantities beyond these amounts bring about decrease in growth and zygote formation. 2. Indications are that the potato extract used contains at least two factors which influence development of Phycomyces. One factor can be adsorbed on charcoal and is freed from it in the presence of ammoniacal acetone. This factor is designated at present Z1. The other factor is not adsorbed to any great extent on charcoal and following filtration is present in the filtrate. This factor is designated Z2. 3. Either of the factors, when used in the relatively impure state described here, produced some stimulative effect upon development, but a combination of the two stimulated development far more than either used alone. 4. A concentrate of the anti gray factor obtained from Dr. Agnes Morgan stimulated growth of Phycomyces when used alone in solution I but was much more effective when used in combination with the fraction containing the Z2 factor. This may indicate that the anti gray concentrate is especially rich in the Z1 factor. 5. The Z1 factor may be identical with biotin. The properties of the Z2 factor have not as yet been correlated with those of any other known growth factor or vitamin.

Growth Substances and Gametic Reproduction by Phycomyces

At 25⚬ C. the intensity of gametic reproduction of Phycomyces blakesleeanus upon an agar medium containing dextrose, asparagine, mineral salts, and thiamin was affected by the distance between the inocula of the plus and minus strains, by the concentration of the agar, by the addition of plant extracts, by the amount of asparagine, and to a less extent by the amount of carbohydrate. Gametic reproduction was decreased with the greater distance between the inocula, and by the larger amounts of asparagine; it was favored by increasing agar concentration and by the addition of plant extracts. The beneficial materials in agar could be extracted in part by dilute methyl alcohol or by aqueous pyridine. The addition of plant extracts overcame in part the inhibitory effect of the larger amounts of asparagine. Little gametic reproduction was obtained at 25⚬ C. in a liquid medium containing dextrose, asparagine, mineral salts, and thiamin unless a plant extract was added. It is concluded that growth substances in addition to thiamin are probably concerned in the gametic reproduction of Phycomyces.

Direct Assimilation of Organic Carbon by Ceratodon Purpureus

1. Under the conditions of the experiments reported organic carbon in the form of levulose, glucose, galactose, lactose, cane sugar, and maltose is absorbed and utilized by Ceratodon purpureus. 2. Starch is formed in the dark from levulose, glucose, galactose, lactose, cane sugar, and maltose. 3. Mannite, glycerine, and starch cannot be utilized by this moss. 4. The amount of growth with levulose as the source of carbonis 2-7 times greater than that with glucose as the source of carbon. 5. In the presence of levulose the greater amount of growth occurs in the dark. With glucose the greater amount of growth occurs in the light. 6. Light seems to be necessary for the formation of moss plants, even though available carbohydrate is furnished.

Further Observations on Factor Z

1. Methods of estimating factors Z1 and Z2 in solutions of unknown composition are described. 2. Neopeptone was found to be rich in Z1 and less rich in Z2. 3. A pyridine extract of Difco agar was found to contain factor Z1 but little Z2. This extract influenced the mature dry weight of Phycomyces in the presence of excess thiamin. 4. The cathode liquor from shredded agar contained Z1 but little or no Z2. The anode liquor contained neither. 5. Z1 was not found to be identical with biotin, pantothenic acid, glutamin, or para-amino benzoic acid. Z2 was not found to be identical with glutamin or para-amino benzoic acid.

GROWTH REQUIREMENTS OF DERMATOPHYTES

It is not my intention to discuss the growth requirements of the dermatophytes in general, but to confine my attention chiefly to two with which we have been mainly concerned. One of these is Trichophyton discoides; the other is Trichophyton mentagrophytes. The strain of T. discoides with which we worked was obtained from Dr. Juan Mackinnon of the Institute of Hygiene, Montevideo, Uruguay. It was found that for growth this isolation required the presence of molecular thiamine, pyridoxine, and inositol in the medium. It failed to grow on a medium which was limited to dextrose, mineral salts, and asparagine. The addition of thiamine, pyridoxine, and inositol to this basal medium permitted considerable growth to occur. All three vitamins were necessary. Little or no improvement in growth was obtained with the addition of any one of the three or any two of the three. The thiamine deficiency could not be satisfied by the pyrimidine component of thiamine, by thiazole, or by a mixture of the two thiamine intermediates. Neither calcium phytate nor a phosphatide containing inositol obtained from Dr. D. W. Woolley were as satisfactory as inositol. The phosphatide substituted for inositol more nearly than did the calcium phytate. The minimum effective quantities of the three vitamins were not determined, but the addition of 0.01 mg. of inositol to 8 ml. of medium which contained both thiamine and pyridoxine produced a marked effect on growth. The maximal effect was obtained with between 0.1 and 0.5 mg. of inositol. The addition of 0.001 mp mole of pyridoxine to 8 ml. of medium containing thiamine and inositol was ineffective, but 0.01 rnp mole produced a measurable effect. Maximum growth was obtained with between 1 and 10 mp moles of pyridoxine. The addition of 0.01 mp mole of thiamine to 8 ml. of medium containing pyridoxine and inositol markedly increased growth, and the maximum effect was obtained with between 1 and 10 mp moles of thiamine. Neopeptone in the amounts we used did not contain sufficient thiamine for maximum growth. Better growth was obtained when the mineraldextrose-peptone medium was supplemented with thiamine. Inositol or a physiological equivalent material was found to be present in hydrolyzed gelatin, hydrolyzed casein, hydrolyzed egg albumen, and peptone, as evidenced by the growth of the organism on these media supplemented with thiamine and pyridoxine but no inositol. Although it was possible to obtain considerable growth on the basal medium of mineral salts, dextrose, and asparagine, supplemented with thiamine, pyridoxine, and inositol, better growth was obtained on a thiamine peptone medium or on gelatin hydrolysate or casein hydrolysate supplemented with the necessary vitamins. It appeared, therefore, that gelatin

Mineral Oil and Preservation of Fungous Cultures

(1949). Mineral Oil and Preservation of Fungous Cultures. Mycologia: Vol. 41, No. 6, pp. 632-636.

Light and the development of Poria ambigua.

In the course of investigations on the nutrition of some of the Basidiomycetes, it was noted that a culture of Poria ambigua Bres. from Ross W. Davidson, his number 86357, formed basidiospores in the light but produced few or none in the dark. The influence of light on the development of many fungi is well known and an extensive literature on the subject exists which will not be reviewed here. Our preliminary observations indicated that Poria ambigua in its response to light might be an especially favorable subject for investigation. Methods and Materials.-The basal medium 1 employed contained mineral salts, dextrose, casein hydrolysate, various purine and pyrimidine bases and the known B vitamins. The hydrion concentration was approximately pH 4.5. Cultures were grown in triplicate or quintuplicate at 25? C in 20 x 100 mm petri dishes containing 20 ml of medium, solidified with 1.5 per cent Difco agar or in 125 ml Erlenmeyer flasks which contained 25 ml of liquid medium. Our standard inoculum was obtained from colonies which had grown three or four days at 25? in the dark. Inoculum, unless otherwise stated, consisted of bits of mycelium 1 mm or 5 mm in diameter taken 3 or 4 mm from the edge of the colony by the method described by Yusef (1953). After inoculation, the dishes were wrapped individually in aluminum foil and incubated in a light-tight incubator at 25?. Unless otherwise indicated, the light to which cultures were exposed was a mixture of diffuse daylight and artificial light totaling from 200 to 280 foot-candles (ft-c). Natural Materials and Growth.-Poria ambigua grew in our basal medium. However, growth of the fungus as measured by dry weight