Part 2
8 0.1 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 1 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 1 percent glucose. Series 74. Ammonium sulphate (0.621 gr. per liter), with 1 percent glucose. ° Series 7B. Ammonium sulphate (as above), with 1 percent mannite. Series 8. Ammonium nitrate (0.5 gr. per liter)—no glucose or mannite. Series 8A. Same solution, with 1 percent glucose. Series 8B. Same solution as series 8, with I. percent mannite. Series 9. Calcium nitrate [Ca(NOs)2.4H:20, 1.475 gr. per liter|—no glucose or mannite. Series 9A. Same solution, with 1 percent glucose. Series 9B. 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 were cleaned in the usual way and dried in the hot airoven. 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. 1, 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 1 mg. for the amount present in each culture flask. It will be noticed that the analyses for th2 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 1n 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 weighing. 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 outerarm. 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 1917-18 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 uninoculated 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. Aeratton.—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. ‘6 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, 200x 25 mm., and containing 25 cc. of standardized N/1o 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 1. Two gas-washing bottles, A and B, TExT Fic. 1. Detail of a portion of one of the series of 1917-18. 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-