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99

AMERICAN

JOURNAL OF BOTANY

OFFICIAL PUBLICATION OF THE BOTANICAL SOCIETY OF AMERICA

EDITORIAL COMMITTEE

C. E. Allen, Editor-in-Chief

University of Wisconsin ^

William Crocker, L. R. Jones,

University of Chicago University of Wisconsifi

C. Stuart Gager, Business Manager Arthur H. Graves,

Brooklyn Botanic Garden Brooklyn Botanic Garden

R. A. Harper, Jacob R. Schramm,

Columbia University Cornell University

C. L. Shear, Bureau of Plant Industry (Representing American Phytopathological Society)

VOLUME VIII1921

WITH TWENTY-SIX PLATES AND NINETY-FIVE TEXT FIGURES

PUBLISHED

IN COOPERATION WITH THE BOTANICAL SOCIETY O

BY THE

BROOKLYN BOTANIC GARDEN At 41 North Queen Street, Lancaster. Pa,

PRESS OF IHE NEW ERA PRINTING COMPANY LANCAbTER, FA.

TABLE OF CONTENTS, VOLUME VIII, 1921

No. I, January

Page

The fixation of free nitrogen by green plants (with one text figure and Plate I) Frank B. Wann i

Variations in the osmotic concentrations of the guard cells during the opening and closing of stomata (with seven text figures)

R. G. WiGGANS 30

The Linnaean cor cept of pearl millet Agnes Chase 41

The effect of cL idiness on the oxygen content of water and its sig- nificance ir cranberry culture (with three text figures)

H. F. Bergman 50

No. 2, February

Influence of temperature on the relations between nutrient salt pro- portions and the early growth of wheat W. F. Gericke 59

The vascular anatomy of dirnerous and trimerous seedlings of Phaseolus vulgaris (with twenty-three text figures)

J. Arthur Harris, Edmund W. Sinnott, John Y. Pennyp acker,

and G. B. Durham 63 Aspergillus flavus, A. oryzae, and associated species (with one text figure) Charles Thom and Margaret B. Church 103

No. 3, March

A study of Rhus diversiloba with special reference to its toxicity (with

two text figures and Plate II) James B. McNair 127

The effect of salt proportions and concentration on the growth of

Aspergillus niger (with six text figures) C. M. Hafjnseler 147

Suggestions with respect to the measurement of osmotic pressure

(with one text figure) L. Knudson and S. Ginsburg 164

Thick-walled root hairs of Gleditsia and related genera (with three

text figures) W. B. McDougall 171

A simple method for growing plants (with one text figure)

J. M. Brannon 176

No. 4, April

The morphology and anatomy of Rhus diversiloba (with Plates III

and IV) James B. McNair 179

iii

iv

TABLE OF CONTENTS

Distribution of the Malvaceae in southern and western Texas (with

one text figure) Herbert C. Hanson 192

Note on the histology of grain roots (with four text figures)

Grace A. Dunn 207 North American Pipers of the section Ottonia (with Plates V-VHI)

William Trelease 212 Monocarpy and pseudomonocarpy in the cycadeoids (with one text

figure and Plates IX-XH) G. R. Wieland 218

No. 5, May

Isoachlya, a new genus of the Saprolegniaceae (with Plates XHI

and XIV) C. H. Kauffman 231

The transmission of Rhus poison from plant to person

James B. McNair 238

The type concept in systematic botany. . . A. S. Hitchcock 251

The relation of certain nutritive elements to the composition of the

oat plant (with two text figures) James Geere Dickson 256

No. 6, June

Specialization and fundamentals in botany. .Joseph Charles Arthur 275 Certain aspects of the problem of physiological correlation

C. M. Child 286 Water deficit and the action of vitamines, amino-compounds, and

salts on hydration D. T. MacDougal 296

The eusporangiate ferns and the stelar theory (with seven text figures)

D. H. Campbell 303

The relation of plant pathology to human welfare F. L. Stevens 315

No. 7, July

The relation of crop-plant botany to human welfare

Carleton R. Ball 323 Correlations between anatomical characters in the seedling of Phaseolus vulgaris (with eight text figures)

J. Arthur Harris, Edmund W. Sinnott, John Y. Pennypacker,

and G. B. Durham 339 A quantitative study of the effect of anions on the permeability of

plant cells H (with one text figure) Oran L. Raber 366

The mechanism of root pressure and its relation to sap flow

James Bertram Overton 369

No. 8, October

The vascular anatomy of hemitrimerous seedlings of Phaseolus vulgaris J. Arthur Harris, Edmund W. Sinnott, John Y. Pennypacker,

and G. B. Durham 375

TABLE OF CONTENTS

V

The effect upon permeability of polyvalent cations in combination with

polyvalent anions (with one text figure) Oran L. Raber 382

The floral anatomy of the Urticales (with Plates XV-XXII).

Albert Reiff Bechtel 386

Genetic evidence of aberrant chromosome behavior in maize endo- sperm (with one text figure) R. A. Emerson 411

No. 9, November

The interrelationship of the number of the two types of vascular bundles in the transition zone of the axis of Phaseolus vulgaris (with two text figures)

J. Arthur Harris, Edmund W. Sinnott, John Y. Pennypacker,

and G. B. Durham 425 Area of vein-islets in leaves of certain plants as an age determinant

(with Plate XXIII) M. R. Ensign 433

Unusual rusts on Nyssa and Urticastrum (with six text figures)

E. B. Mains 442 Miscellaneous studies on -the crown rust of oats (with Plate XXIV)

G. R. HoERNER 452

Comparative studies on respiration XVIII. Respiration and antagonism in Elodea (with two text figures) C. J. Lyon 458

The effect upon permeability of (I) the same substance as cation and anion, and (II) changing the valency of the same ion (with two text figures) Oran L. Raber 464

No. 10, December

Pollen and pollen enzymes Julia Bayles Paton 471

The embryogeny of Cyrtanthus parviflorus Baker (with Plates XXV

and XXVI) Wm. Randolph Taylor 502

Studies on plant cancers III. The nature of the soil as a determining

factor in the health of the beet, Beta vulgaris, and its relation to

the size and weight of the crown gall produced by inoculation

with Bacterium tumefaciens (with nine text figures)

Michael Levine 507

Index to Volume VIII 526

(Dates of publication: No. i. Mar. 9; No. 2, Mar. 19; No. 3, Apr. 3; No. 4, Apr. 30; No. 5, May 24: No. 6, June 30; No. 7, Aug. 31; No. 8, Nov. 14; No. 9, Dec. 19; No. 10, Feb. 15, (1922.)

vi

ERRATA, VOLUME VIII

ERRATA, VOLUME VIII

Page 31, table I: Eryngium campestra should be Eryngium campestre. Page 145, 7th line should read:

26. Hooker, W. J. Flora boreali-americana i: 126, 127. London, 1833.

8th and 9th lines should read:

27. Hooker, W. J., and Arnott, G. A. W. The botany of Captain Beechey's

voyage, part 3, p. 137. London, 1841. 28th line should read: Lyon, W. S. The flora of our southwestern archipelago II. Bot. Gaz. 11

330-336. 1886. Page 146, 9th line should read: London, 1838. In line 14:

New Yor should be Nezv York. Page 195, line 26: Malva parvifolia should be Malva parviHora. Page 231, 1st line of text

After Kauffman, add and Coker Page 274, 3d line should read:

Wolff, E. 1871. Aschen Analysen 1 : 1-149; 2: 1-170. Page 451, 1 8th line: Urticastri should be Dicentrae

VM. VIII JANUARY, 1921 No. I

AMERICAN

JOURNAL OF BOTANY

OFFICIAL PUBLICATION OF THE BOTANICAL SOCIETY OF AMERICA

MAR 15 1921

CONTENTS

The fixation of free nitrogen by green plants Frank B. Wann i

Variations in the osmotic concentrations of the guard cells during the opening and closing of stomata R. G. Wiggins 30

The Linnean concept of pearl millet . Agnes Chase 41

The effect of cloudiness on the oxygen content of water and its significance in cranberry culture . H. F. Bergman 50

PUBLISHED

IN COOPERATION WITH THE BOTANICAL SOCIETY OF AMERICA At 41 North Queen Street, Lancaster, Pa.

BY THE

BROOKLYN BOTANIC GARDEN Brooklyn, N. Y.

Entered as «ecDnd-cIa»« matter February 21, 1914, at the post office at Lancaster, Pennsylvania, under the act of March 3, 1879

AiMERICAN

JOURNAL OF BOTANY

Devoted to All Branches of Botanicaj. Science

Established 1914

EDITED BY A COMMITTEE OF THE BOTANICAL SOCIETY OF AMERICA

EDITORIAL COMMITTEE

C. E. Allen, Editor-in-Chief, University of Wisconsin

William Crocker, L. R. Jones,

University of Chicago University of Wis . onsin

C. Stuart Gager, Business Manager Orlaxd E. White,

Brooklyn Botanic Garden Brooklyn Botanic Garden

R. A. Harper, Jacob R. Schramm,

Columbia University Cornell University

C. L. Shear, Bureau of Plant Industry (Representing American Phytopatliological Society)

The Journal is published monthly, except during August and September. Subscription price, J6.00 a year. Single copies 75 cents. Back numbers, 75 cents each ; $6.00 a volume, postage extra. Postage will be charged to all f oreign countries, except Mexico, Cuba, Porto Rico, Panama Canal Zone, Republic of Panama, Hawaii, Philippine Islands, Guam, Samoan Islands, and Shanghai. Postage to Canada, 20 cents a volume on annual subscriptions ; to all other (Countries in the Postal Union, 40 cents a volume on annual subscriptions.

The pages of the Journal are open to members of !the Botanical Society of America, or to candidates approved for membership.

Mantiscript offered for publication should be typewritten, and should in all cases be submitted to the Editor-in-Chief.

Papers are limited to 20 pages in length, exclusive of plates, except that ad- ditional pages may be arranged for at cost rate to authors (approximately $3.00 a page).

Proofs should be corrected immediately on receipt, and returned to American Journal of Botany, Brooklyn Botanic Garden, Brooklyn, N. Y.

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Correspondence concerning editorial matters should be addressed to Prof. G. E. Allen, University of Wisconsin, Madison, Wisconsin. ^

Business correspondence, including notice of change of address, and directions concerning reprints, should be addressed to American Journal of Botany, Brook- lyn BiDtanic Garden, Brooklyn, N. Y., or 41 North Queen Street, Lancaster, Pa.

AMERICAN

JOURNAL OF BOTANY

Vol. VIII January, 1921 No. i

THE FIXATION OF FREE NITROGEN BY GREEN PLANTS

Frank B. Wann (Received for publication July 3, 1920)

The ability of chlorophyll-bearing plants to utilize the uncombined nitrogen of the atmosphere has been repeatedly investigated during the last three or four decades, and the results of numerous observations and experiments, covering a wide range of species, have been quite conflicting. In some of the earlier experiments with higher plants in pot cultures the beneficial effect of a surface layer of algae was often observed, and the ability to increase the nitrogen content of the soil by free nitrogen fixation was ascribed to members of both the blue-green (Cyanophyceae) and grass- green (Chlorophyceae) algae. Similar increases in soil-nitrogen content were observed when higher plants were excluded from the cultures, and, though bacteria were known to be present, the fixation was generally ascribed to the chlorophyll-containing forms. More recently, pure cultures of members of the Chlorophyceae have been used but the results in these cases have been almost uniformly negative. This fact, together with the discovery of widely distributed soil bacteria of the Azotobacter and Clostridium types, the ability of which to fix free nitrogen can not be ques- tioned, has led to the belief th^at in impure cultures fixation is due not to the activities of the green plants but to the bacteria present in the soil. Thus it has come to be very generally accepted that members of the Chloro- phyceae, as well as the higher plants, are not able to use free nitrogen.

However, the number of species which have been investigated in pure culture is small, and the culture methods employed have not always been those which are most favorable for the best growth of these organisms. Accordingly the experiments reported here were undertaken for the purpose of extending the observations over a larger number of species, grown on a variety of mineral nutrient solutions under culture conditions which would insure a rapid and vigorous growth.

Literature

A complete, and in some cases detailed, review of the literature bearing on the relation of the grass-green algae (Chlorophyceae) to free nitrogen is available in a paper by Schramm (1914 a), so that a repetition of the account is unnecessary here. Nothing of importance relating to this subject has [The Journal for December (7: 409-468) was issued January 12, 192 1.]

I

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AMERICAN JOURNAL OF BOTANY

[Vol. 8

appeared, so far as the author is aware, since the above-cited paper. As has already been indicated, the results of experiments with pure cultures have been pretty generally negative as regards the ability of these forms to increase the nitrogen content of the culture. In the light of results pre- sented here, some of the previous experimental work will be considered in the general discussion to follow.

Methods

Seven species of Chlorophyceae were isolated and used in pure culture. In making the isolations the plate culture method, as described by Schramm (1914 h), was employed. With the exception of one species, Protococcus sp., all isolations were made from growths occurring on soil; the material for the Protococcus culture was secured from the bark of an elm tree.

The absolute identity of all the species has not as yet been determined. Cultures have been submitted to several authorities on the group, but for some of the forms the determinations received have been somewhat at variance. For that reason no attempt has been made to apply specific names to all the organisms. Moreover, as will be apparent later, the abso- lute identity of the forms, though highly desirable, does not become of paramount importance because of the very similar way in which all the species seem to react. Unless otherwise indicated, therefore, the different species will be referred to by number and genus, or by number only, and as soon as more satisfactory determinations can be made a list will be pub- lished, if possible, in this journal. The forms used in the experiments include the following:

Species number i . Chlorella vulgaris Beyr. There seems to be no doubt about the identity of this species.

Species number 2. Stichococcus sp.

Species number 3. Protosiphon hotryoides (Kg.) Klebs.

Species number 5. Chlorella sp. A small form with cup-chaped chro- matophore.

Species number 6. Scenedesmus sp.

Species number 7. Protococcus sp.

Species number 11. Chlorella sp. A large form with clathrate chro- matophore.

All these species have been carried along on mineral nutrient agar for two or three years; they have been repeatedly transferred to media con- taining glucose or sucrose, and have frequently been examined micro- scopically. They are known to be free from bacteria and are pure cultures in the strict sense.

Culture Media. In the experiments Kjeldahl flasks of Pyrex glass and of 500 cc. capacity were used as culture flasks, because of the obvious advantage of analyzing checks and cultures without transferring the material to a digestion flask. Approximately 150 grams of mineral nutrient agar were supplied as a medium for each culture. In spite of the difliculties

Jan., 1921]

WANN FIXATION OF FREE NITROGEN

3

involved in its analysis, a solid medium was chosen because of the very long-continued, vigorous growth produced on it. So far as has been observed, solution cultures, at least when unaerated, do not give a very extended or abundant development of these organisms. Since many pre- vious experiments have also shown that these forms do not grow in pure culture in the complete absence of combined nitrogen, no attempt was made to include such cultures in the experiments.

Two experiments were performed, the first in the winter of 191 7-18 and the second in the summer of 191 9. In both cases the following mineral nutrient solution was employed as a standard for the preparation of the media:

NH4NO3 0.5 gram

MgS04 0.2 gram

K2HPO4 0.2 gram

CaCl2 0.1 gram

FeS04 trace

Distilled water 1000 cc.

With the nitrogen content of this solution as a basis, the NH4NO3 was replaced in the various series of 191 7-1 8 by glycocoll, asparagine, (NH4)2S04, and CaCNO.Oa, and by urea, (NH4)2S04, and Ca(N03)2 in 1919, the nitrogen content as such being approximately the same in all media. Each of these sources of nitrogen was used in duplicate series, to one of which glucose was added. (In 1919 NH4NO3, (NH4)2S04, and Ca(N03)!> were also used in series to which mannite was added.) No change was made in the other constituents of the full nutrient solution, so that in all series these salts were present in the proportions indicated above. The 191 7-18 experiment included the following series, arranged according to nitrogen sources and presence or absence of glucose :

Series i. Glycocoll (1.07 gr. per liter) no glucose.

Series lA. Same solution, with i percent glucose.

Series 2. Asparagine (0.942 gr. per liter) no glucose.

Series 2A. Same solution, with i percent glucose.

Series 3. Ammonium sulphate (0.942 gr. per liter) no glucose.

Series ^A. Same solution, with i percent glucose.

Series 4. Ammonium nitrate (0.5 gr. per liter) no glucose.

Series 4^. Same solution, with i percent glucose.

Series 5. Calcium nitrate [Ca(N03)2.4H20, 1.475 gr. per liter]— no glucose. Series 5^4. Same solution, with i percent glucose.

In making up the media for the above series, sufficient nutrient solution with any one nitrogen source was prepared to supply both the series without glucose and the series with glucose. For these solutions the required amounts of the several constituents, with the exception of ferrous sulphate, were weighed out individually and dissolved in the proper volume of distilled water. (A stock solution of ferrous sulphate was prepared by dissolving

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AMERICAN JOURNAL OF BOTANY

[Vol. 8

O.I gr. in 2,000 cc. of distilled water, 50 cc. of which were used in the prepara- tion of each liter of nutrient solution.) The solution thus prepared was divided into two equal quantities in large flasks, 1.5 percent agar being added to each, and i percent glucose to one portion. The total nitrogen content of the medium with any one nitrogen source should therefore be the same per unit weight in the two series, with and without glucose, except for traces of nitrogen in the glucose or for slight discrepancies in the actual amount or composition of agar added. The nitrogen-containing compounds were not dried to constant weight nor was the agar purified in any way, all substances being added to the solution directly from the stock bottles. The chemicals used were Baker's "analyzed" and Merck's ''highest purity"; the agar was of the kind known as "Difco bacto."

The total nitrogen content of each culture medium was determined by actual analysis of weighed portions of that medium. It is obvious that by this method any nitrogen introduced with the agar or as impurities with the glucose would be completely accounted for.^

The 1919 experiment was a partial duplication and an extension of that of the previous year and included the following series:

Series 6. Urea (0.375 gr. per liter) without glucose. Series 6A. Same solution, with i percent glucose.

Series yA. Ammonium sulphate (0.621 gr. per liter), with i percent glucose. Series "jB. Ammonium sulphate (as above), with i percent' mannite. Series 8. Ammonium nitrate (0.5 gr. per liter) no glucose or mannite. Series 8^. Same solution, with i percent glucose. Series 8B. Same solution as series 8, with i percent mannite.

Series 9. Calcium nitrate [Ca(N03)2.4H20, 1.475 gr. per liter] no glucose or mannite.

Series gA. Same solution, with i percent glucose.

Series gB. Same solution as series 9, with i percent mannite.

Each complete solution was placed in the autoclav under 15 pounds' pressure until the agar was dissolved. The solution was then filtered through absorbent cotton; the filtrate was free from sediment.

Introduction of the Agar. The Kjeldahl flasks wer-e cleaned in the usual way and dried in the hot air oven. On removal from the oven, cotton plugs were inserted in the mouths of the flasks to prevent the entrance of dust. Each flask was numbered by means of a carborundum point and weighed to within 0.05 gram on a Mackenzie one-pan balance, the cotton plug being removed only during the weighing. The flasks were then stored in clean, dry boxes until required.

As soon as the medium for a series was prepared the required number of flasks (11 in 1917-18, 24 in 1919) were arranged in one of the special wooden racks (see fig. i, Plate I) and 150 cc. of the hot agar solution was added to

1 Numerous analyses of the agar showed the nitrogen content to be about i mg. for the amount present in each culture flask. It will be noticed that the analyses for the total nitrogen of the media may not have yielded exactly the calculated amounts, because of the moisture present in the nitrogen-containing compounds.

Jan., 1921]

WANN FIXATION OF FREE NITROGEN

5

each flask. The flasks were then immediately weighed in the order in which the agar was introduced. The mouths of the flasks remained plugged with cotton except during the actual processes of introducing the agar and of w^eighing. Although water vapor condensed on the walls and necks of flasks as the agar cooled, the use, with a number of flasks, of rubber stoppers instead of cotton plugs, demonstrated that there was no detectable loss of water vapor through the cotton plug during the interval between the intro- duction of the agar solution and the weighing. Thus the actual weight of medium in each flask was known, and, as will be seen from the tables, differences resulted of one or two grams in the weight of approximately equal volumes between the first and last flask of a series to receive the medium, due to the cooling of the agar during the processes involved.

After the second weighing each flask was provided with a two-hole rubber stopper carrying intake and outlet glass tubes, the outer arms of these tubes being adjusted so as to be readily connected in series by means of rubber tubing. Long cotton plugs were loosely adjusted in the bore of each outer arm. The flasks were sterilized at 15 pounds' pressure for 20 minutes, the stoppers resting lightly in the mouths of the flasks but being tightly adjusted upon removal from the autoclav, the hands being moistened with alcohol for this operation. The flasks were allowed to cool in a dust-proof case.

Inoculation. The inoculations were made in the laboratory under a glass dust shield open on one side only. The inoculum consisted of a suspension of the algal cells in a test tube of sterilized nutrient solution minus combined nitrogen. Special cultures on hard (2 percent) agar were pre- pared, so that in making the suspension no agar was introduced with the cells. The tube was thoroughly shaken to secure a uniform suspension of the inoculum, of which ten drops were added to each flask in the 191 7-1 8 experiment and one cubic centimeter was similarly added in 1919. In the former experiment four species were used and two flasks in each series were inoculated with the same species, three flasks of each medium remaining uninoculated as checks. In 1919 seven species were used, three flasks of each series being inoculated with each species with the exception of species no. 3 and no. 7, in which cases only two flasks of any one medium were inoculated; three flasks of each series remained uninodulated as checks.

After the rubber stoppers were tightly fitted in the flasks, melted paraffin was run in around the flared neck, and the stopper and a portion of the neck of each flask were covered with sterilized cotton. During the two experiments eighteen contaminations occurred out of a total of 340 flasks.

Aeration. The intake and delivery tubes of the flasks of each series were connected with rubber tubing for the purpose of aeration. In making connections the free ends of the glass tubes were painted with 95 percent alcohol, which was used also in washing out the bore of the rubber tubing.

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AMERICAN JOURNAL OF BOTANY

[Vol. 8

In setting up the first experiment it was thought that in the process of aeration a loss of nitrogen from the medium in the form of ammonia might occur, especially in view of the fact that the medium was slightly alkaline, so that a tube of acid was inserted in the series just beyond each culture flask. For this purpose large test tubes, 200x25 mm., and containing 25 cc. of standardized N/io sulphuric acid were used. As a precaution against the backflow of this acid into the cultures, small Erlenmeyer flasks of 180 cc. capacity were placed between each culture flask and its corre- sponding acid tube. The arrangement of the apparatus can readily be understood by consulting text figure i. Two gas-washing bottles, A and B,

Text Fig. i. Detail of a portion of one of the series of 191 7-1 8. Explanation in the text.

were placed at the head of each series; A contained 30 percent sulphuric acid and a quantity of pumice stone; B contained sterilized distilled water. Air entered the series through a calcium chloride tube filled with cotton, was washed free of ammonia by the acid in A and was moistened by the water in B. Oxides of nitrogen would also be removed by the water. Before entering the culture flask C the air passed through a second calcium chloride tube containing sterilized cotton. The intake tube of each culture extended to within an inch or so of the surface of the agar medium, whereas the delivery tube merely penetrated the rubber stopper, so that in the process of aeration the air above the agar surface was completely changed. After leaving the culture flask, the air passed through the safety flask D' and bubbled through the acid in the adjoining test tube E'. The intake tube of the latter was drawn out to a fine point which extended to the very bottom of the test tube, so that only very small bubbles were formed, insuring a thorough washing of the air before it passed into the next culture flask of the series. The delivery tube of the last acid tube in the series was connected to a filter pump, by means of which the air was drawn through the whole series at once.

When the ten series of the first experiment had been completely as-

Jan., 1921]

WANN FIXATION OF FREE NITROGEN

7

sembled, the delivery tubes at the end of each rack were connected in a single series by means of T-tubes so that aeration of all ten series could be accomplished by one operation. Each of these delivery tubes was provided with a screw clamp which was kept tightly closed except during the process of aeration, when it served to control the volume of air passing through the series. With a few exceptions aeration was continued for an hour every morning, it being considered that this would entirely replace the air in the apparatus. The 191 7-1 8 experiment as completely assembled is shown in figure I, Plate I.

At the end of the first experiment, titrations of the contents of the acid tubes showed that no appreciable change had taken place in the concentra- tion in any instance. It was assumed, therefore, that with these species and with the conditions realized in the experiment there was no loss of ammonia from the culture flasks. For this reason the tubes of acid were omitted from the second experiment, and in the process of aeration the air passed directly from one culture flask to the next in the series. Because of the expansion of the air in the culture flasks during the hot summer days, the liquids in the gas-washing bottles were frequently forced out through the intake tubes of these bottles, making it necessary to place safety flasks outside the acid bottle, and between the acid and water bottles, to re- ceive those liquids. During the process of eration the acid and water were drawn back into the proper bottles so that no air ever entered the series without first passing through the liquids. Aeration was continued for an hour every other morning during the growing period.

Cultural Conditions. Soon after the inoculation of the culture flasks the ten racks were transferred to the greenhouse where more uniform condi- tions of light and temperature prevailed than in the laboratory. Since preliminary tests showed that agar cultures of the organisms used were soon killed by direct sunlight, the bench occupied by the apparatus was covered with a canopy of black cloth, which reduced the actinic light intensity to about one eighth that of the normal greenhouse illumination. On cloudy days, however, this canopy was rolled up on both sides, thus permitting better illumination ; on clear days the west side was open during the morning only, and during the afternoon the east side only was exposed. The arrangement of the apparatus as finally assembled in the greenhouse is shown in figure 2, Plate I, which is a photograph of the 191 9 experiment.

Growth of the Cultures

Length of Growing Period. The approximate length, in days, of the growing period of each series is indicated in the headings of the tables which follow. Inoculations in the first experiment were completed on August 31, 191 7, and the analyses were begun in April, 191 8. The final inoculations of the second experiment were made in May, 191 9, and analyses were started in November of the same year.

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AMERICAN JOURNAL OF BOTANY

[Vol. 8

Method of Recording Growth. Records of the growth of the cultures were made at intervals of three or four weeks. These consisted of written notes comparing the development of the different species on the same medium, and the difference in amount of growth of the same species on the ten different media. Charts were also prepared at about monthly intervals showing the growth in each culture flask by means of colored crayons. These were found very helpful in making growth comparisons, as they present to the eye at once the relative development in every flask.

Since it was the plan of the experiments to analyze the entire contents of the culture flasks at the end of the growing period, it was not found advisable to attempt any actual weight determinations of the ''crop" produced on the various media by the different species. Experiments with solution cultures are in progress now from which it is hoped some accurate data may be secured, showing the actual amounts of growth produced on different media and what relation the dry weight of algal material produced bears to free nitrogen assimilation. From a comparison of the written notes and colored charts, however, the following general statements may be made.

igiy-i8 Experiment. In this experiment there was a remarkable sim- ilarity in the amount of development of all four species on any one of the media used. Only in a few cases did there appear to be any marked specific differences in the reactions of the organisms to the medium. In general, the presence of glucose resulted in a vigorous and rapid development of all species, irrespective of the nitrogen source.

Series i-j. The relative growth of all the cultures is indicated in the tables which follow, by means of plus signs. Since no fixation occurred in the series in which the nitrogen was supplied as ammonium sulphate or in the organic forms used, the detailed observations of these series are omitted. The results on the nitrate media, however, were so striking that a more detailed account of the growth on these media is here presented.

Series 4 (ammonium nitrate, without glucQse). Growth was slow but steady in all cases. Species nos. i, 5, and 6 continued healthy to the last, giving "very fair" growths. The growth of species no. 2 was "fair," but the cultures were dead at the end of the experiment.

Series 4A (ammonium nitrate, with i percent glucose). All species started with very vigorous growths, the effect of the presence of glucose being very evident. Species no. i produced a "luxuriant" growth at first, but after three months began to deteriorate, turning brown over most of the surface. Before growth had completely ceased, however, both cultures of this species began to revive, and by the end of the fourth month were again bright green. The cultures then, slowly waned a second time, only small portions remaining green at the end of the experiment. Species nos. 2, 5, and 6 grew steadily from the start and remained healthy, no. 2 producing a "luxuriant" growth and nos. 5 and 6 "very good" growths.

Jan., 1921]

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Series 5 (calcium nitrate, without glucose). The growth was very slow with all species, but all remained healthy. Total growth of species no. 2 was ''fair," while for nos. i, 5, and 6 it was ''very fair."

Series (calcium nitrate, with i percent glucose). All four species gSive vigorous growths on this medium, continuing healthy to the end of the experiment. The effect of the presence of glucose was apparent from the start. The growth of species no. 2 was "luxuriant" and appeared slightly better than the others, all of which were "very good."

igip Experiment. So^ far as this experiment duplicated the previous one, the same general type of growth resulted. The presence of glucose in the medium markedly stimulated the development of all species, irre- spective of the source of combined nitrogen. It is also true, however., that death of the cultures always occurred first on the media containing glucose. The presence of mannite apparently had no effect by way of increasing the rapidity or amount of growth of any of the species on any of the three media to which this compound was added. The amount of growth produced on these media appeared practically the same as produced by the same organism with the same source of nitrogen but without either glucose or mannite.

Series 6 and y. As in the previous experiment, no fixation occurred where combined nitrogen was supplied in an organic form or as ammonium sulphate; growth observations for these media are therefore omitted.

Series 8 (ammonium nitrate, without glucose or mannite). A slow, steady growth resulted, as in the previous experiment. At analysis all cultures were healthy. Species nos. i, 6, and 11 produced "very fair" growths; nos. 2 and 5, "fair." Species nos. 3 and 7 were not grown on this medium.

Series 8 A (ammonium nitrate, with i percent glucose). All species grew very vigorously at first, but deterioration soon set in and by the end of one month all' cultures of nos. i and 2 were dead, after a "fair" growth, and species nos. 3 and 6 were rapidly waning. The cultures of no. 3 died after a "fair" growth. One culture of no. 6 also died, but the two others revived and remained healthy to the end of the experiment, giving "very good" growths. Species no. 11 gave a "luxuriant" growth, but at the end of the experiment was turning brown. Nos. 5 and 7 remained healthy throughout the growing period, both producing "very good" growths.

Series 8B (ammonium nitrate, with i percent mannite). Growth on this medium was slow, and in general very much as in series 8. All cultures remained healthy at the end of the experiment. Species nos. i, 3, 6, and II gave "very fair" growths, the development being somewhat better than with species nos. 2, 5, and 7.

Series g (calcium nitrate, without glucose or mannite). Growth was very slow, and strikingly like that in series 8. All cultures remained healthy, species no. 6 giving a "good" growth, nos. i and 11 "very fair"

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growths, and nos. 2 and 5 "fair." Species nos. 3 and 7 were not included in this series.

Series gA (calcium nitrate, with i percent glucose). Development was very vigorous from the start, all cultures soon producing a complete coat over the agar surface. At the end of a month, species nos. 3, 5, 6, and il began to turn yellow. The deterioration, however, did not progress far, and after another month or two all cultures were again green, remaining healthy until analysis. Species nos. 1,2, and 7 remained healthy throughout the entire growing period. At the end of the experiment a "luxuriant" growth had resulted with species nos. 2 and ii, "very good" growth with nos. I, 5, and 6, "good" with no. 3, and "slight" with no. 7.

Series gB (calcium nitrate, with i percent mannite). The development was very similar to that in series 9, being slow but steady. All cultures remained healthy to the end. Species nos. i and 11 produced "very fair" growths, nos. 2, 5, 6, and 3 "fair," and no. 7 only "slight."

It should be noted that in the 191 7-1 8 experiment the development of the four species on the two nitrate media with glucose (series and 5^) was very similar, the growth being either "luxuriant" or "very good" in all cases. Of the two, however, the cultures of series 5A appeared some- what the better. In striking contrast with this condition was the growth of the various species on similar media of the 1919 experiment (series 8^ and ()A). The species which gave such good growths on ammonium nitrate with glucose (series 4^) in 191 7-1 8 showed scarcely any develop- ment on this medium in 191 9 (series 8^4), with the possible exception of species no. 5. However, on calcium nitrate with glucose (series 9^) the growth of these species was practically the same as was secured on the similar medium of I9i'7-i8 (series 5^). Even though a few of the flasks of series 8^ were reinoculated, the growth continued poor; likewise, "reserve" flasks of this medium which were not introduced in the series at the beginning of the experiment gave very similar growths of species nos. I and 2 when inoculated a few months before the end of the growing period. This difference in the amount of growth produced in the two experi- ments may possibly be related to the difference in the seasons during which the experiments were conducted.

Analyses

At the end of a growing period of from six to eight months the cultures and checks were analyzed for total nitrogen content. After making a final record of the growth and condition of the cultures, a small loop of the algal material from each flask was transferred to a drop of sterilized nutrient solution and examined microscopically. In 191 8 transfers were also made from each culture to nutrient agar containing i percent glucose, and to the same medium made acid by the addition of I percent hydrochloric acid. Two tubes of each of these media wete inoculated from each culture flask, but in no case did contaminations appear that were not apparent from the

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microscopical examination. This precaution was therefore omitted in 1919, the microscopical examination being relied upon to detect contaminations not apparent to the naked eye. In all cases, however, such contaminations as occurred were perfectly evident from macroscopic examinations, the majority of them appearing soon after inoculation.

After considerable prehminary work with total nitrogen determinations, the Gunning-Kjeldahl method was adopted for media free from nitrates, and in the presence of nitrates the Forster modification of this method was used. In the former method the digestion mixture consists of a solution of 20 grams phosphorus pentoxide (P2O5) in 500 cc. sulphuric acid of sp. gr. 1.84, 20 cc. of this solution being added to each culture flask. After the addition of 10 grams potassium sulphate to each, the flasks were heated slowly over a low flame. The presence of the agar made the digestions particularly trying, as it was necessary to watch the flasks constantly at the beginning of the process in order to prevent the contents from foaming up into the necks. It was found advisable to start with a very low flame and to shake the flasks occasionally until sufficient agar had gone into solution to allow the ready escape of bubbles from below. At this point the full flame was used, and the water boiled off vigorously until foaming began. The flame was then turned very low again for about 30 minutes or until the appearance of dense, white fumes, at which time foaming gradually ceased. The fire was then slowly increased to full capacity and the digestion con- tinued for 15 or 20 minutes after a clear liquid resulted. About I J hours were required for digestion after foaming ceased. In all cases the flasks at the end of the digestion were perfectly clean and the liquid was entirely transparent.

The distillation was carried out in the usual way. About 150-200 cc. of distilled water was added to each flask when cool, the neck of the flask being thoroughly washed down in the process. The solution was made alkaline with 50 cc. concentrated sodium hydroxide, and, after the addition of a gram of granulated zinc, the flask was immediately connected to the still. The ammonia was distilled over through block tin tubes into a 500-cc. Erlenmeyer flask containing 30 cc. standardized tenth-normal sulphuric acid, diluted with enough distilled water to cover completely the end of the delivery tube. Standard traps were used between the Kjeldahl flask and the condenser. Distillation was continued until the contents of the flask began to "bump." About 125 cc. of water was distilled over in this process, and it was usually completed in about 40 minutes. During the latter part of the distillation the receiving flask was drawn away from the still sufficiently to uncover the end of the delivery tube, the inside walls of which were washed down by the remaining distillate. At the end of the distillation the delivery tube, as well as the inside wall of the receiving flask, was washed down with a small amount of distilled water. The excess acid was titrated against tenth-normal sodium hydroxide, using cochineal as an indicator.

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The Forster modification, employed in analyzing the media containing nitrates, consists essentially in the addition of sodium thiosulphate to the digestion mixture. The procedure was as