Quantitative Studies on the Ciliate Glaucoma. Excretion of Ammonia

Griffiths (1888) used mass cultures of Amoeba, Paramecium and Vorticella and concluded that the secretions of the contractile vacuole are capable of yielding microscopic crystals of uric acid.

Howland (1924) found that uric acid is formed in old cultures of Amoeba and Paramecium in hay infusions and inferred that uric acid is excreted by these organisms.

Weatherby (1927) concluded that in Paramecium at least some of the nitrogen is excreted in the form of urea, and that ammonia formed in cultures arises from the hydrolysis of urea by the bacteria present.

Weatherby (1929) made tests for ammonia, urea, uric acid and creatine in a nonnutritive medium in which previously washed specimens of Paramecium, Spirosto-mum and Didinium had been kept. He concluded that Paramecium and Spirostomum excrete nitrogen solely as urea and that Didinium excretes nitrogen mostly in the form of ammonia with possibly some uric acid. He found that animals kept in a non-nutritive medium produced nitrogen for a period of 2–5 hours and that thereafter very little was produced. When sufficient food was present nitrogen continued to be produced at the initial rate. After testing the fluid of the contractile vacuoles for urea, he concluded that the contractile vacuole could eliminate only an insignificant fraction.

Lwoff (1932) used pure cultures of Glaucoma piriformis grown in yeast autolysate. He found that, over periods of 6–40 days, the amide and ammonia fraction of the total nitrogen increased at the expense of more complex nitrogenous materials. He found that uric acid and urea were not produced, and that when urea was added it was recoverable quantitatively.

Lawrie (1935) found that Bodo caudatus grown on a bacterium produced ammonia but neither urea nor uric acid, and that urease activity was absent. For the first 8–0 hours of the growth of a culture the rate of ammonia production was found to be proportional to the number of Bodo present. After this time the rate decreased.

In order to relate nitrogenous excretion somewhat more closely to the morphologically observable events occurring in the organism, we have attempted to measure the latter in detail at the same time that the nitrogen was being measured. In order to obtain the required numbers of Protozoa in uniform condition we have employed the methods developed by Harding (1937).

To estimate the amount of nitrogen entering the organism we have determined the nitrogen content of the food material, the rate of formation of food vacuoles, the nitrogen content of the material in a single vacuole and the amount of nitrogen in the food which is in the same form as that eliminated by the protozoan. In order that the sole entrance for nitrogen should be the material in the food vacuoles we have chosen a particulate source of food, viz. the bacterium Pseudomonas fluorescens. This constitutes a completely adequate diet for Glaucoma. The nitrogen excreted was measured at hourly intervals. The rate of respiration, the rate of multiplication, the change in size of organisms, the change in amount of visible food reserves, and the rate of elimination of food vacuoles were either measured directly or estimated from parallel experiments.

The glaucomae were cultured at 25 ± 0·1° C. on suspensions of Pseudomonas and counts were made using the methods previously described (Harding, 1937). It was necessary to use very high concentrations of Glaucoma so that the concentration of nitrogen excreted would be sufficient for analysis. This made it difficult to keep the Glaucoma as fully fed as in previous experiments. They were, however, sufficiently well fed to be multiplying at a rapid rate. In the previous experiments it was found that, under conditions in which Glaucoma form ten food vacuoles per hour, the food vacuoles are eliminated 4 hours after they are formed, and that each Glaucoma eats about ten thousand bacteria per hour. In organisms of the size and condition used in the experiments described below the food vacuoles are 8·0±0·5μ in diameter when formed and each vacuole contains approximately one thousand bacteria. Glaucomae which had been fed in this manner for 12–24 hours were washed free of all but a negligible number of bacteria by centrifuging and transferred to Peter’s solution.

For the estimation of ammonia the method described by Conway & Byrne (1933) was used. 1 ml. samples of the fluid to be analysed for free ammonia were put into Conway cells and distilled from 40 per cent NaOH to N/100 H₂SO₄. An aliquot of the acid was mixed with the Berthelot reagent described by Van Slyke & Hiller (1933) and the colour read against standards of (NH₄)_aSO₄.

In urea estimations, 5 ml. samples were incubated with B.D.H. urease and the ammonia estimated as described above.

Total nitrogen was estimated by combustion with H, SO₄, H₃PO₄, and selenium as described by Borsook (1935). After combustion an aliquot of neutralized solution was analysed as described for free ammonia. We are indebted to N. R. Lawrie for a description of his modifications of Borsook’s procedure.

In determining the experimental procedure it was necessary to know the effect of the bacteria on the culture fluid in relation to nitrogen as well as the total nitrogen content of the bacteria.

A suspension of Pseudomonas prepared as described above was made and immediately tested for urea and free ammonia. Three hours and 24 hours later the same tests were made. No urea was found at any time. The amounts of free ammonia found are given in Table I.

Harding has shown that Peter’s medium is non-nutritive to Pseudomonas, even when the metabolic products of Glaucoma are present, but there is present in the suspension an extract of the substrate on which the bacteria were grown. It is the nitrogen from this source which is utilized by Pseudomonas. Similar findings have been reported previously (Buchanan & Fulmer). The substrate nitrogen entering Glaucoma along with the food was, in the following experiments, a negligible quantity.

Determination of the total nitrogen gave an average value of 0·0125 mg. per 10⁹ bacteria. If this combustion value represents 85–90 per cent of the true total nitrogen, there should be approximately 0·0143 mg. per 10⁹ bacteria.

In all tests on freshly prepared and older cultures with and without bacteria no urea was detectable. This is in agreement with the experiments of Lwoff (1932), who further found that added urea was quantitatively recoverable.

In determining the amount of ammonia produced in initial experiments the ciliates were separated from the culture solution by centrifuging. Parallel determinations were made with the ciliates left in the solution and these gave values identical within the limits of the technique. In later experiments the ciliates were not removed from the solution to be analysed. Culture fluid sterilized by heating was found to maintain a constant NH₃ so that extra precautions to prevent loss of NH₃ were considered unnecessary.

Within 5 min. after the fed glaucomae had been washed free of bacteria and suspended in Peter’s solution, the first samples for ammonia analyses were taken. This zero time value was usually almost unreadable and always less than 0·0005 mg-per ml.

During the time in which the glaucomae were feeding on the bacteria, there was an ammonia concentration in the medium, which was determined by the excretion of the glaucomae and by the utilization of NH₃ by the bacteria. This ammonia entered the food vacuoles with the bacteria. If one assumes that the vacuoles contain only this fluid, viz. that the bacteria occupy none of the volume, then the amount of ammonia available from this source is an entirely negligible fraction of the ammonia excreted after removal of the bacteria. It is conceivable that this free ammonia might be accumulated by the ciliate by other means. This point was tested by leaving starving Glaucoma in an ammonia-rich solution for 24 hours. Upon transfer to Peter’s solution they liberated a negligible quantity of ammonia.

From these results we concluded that all significant amounts of nitrogen entering Glaucoma during the course of the experiments were entering as bacteria.

The results of typical experiments are shown in the tables and graphs.

In Exp. 19 there were approximately 1000 bacteria per food vacuole, and there were 1·19 × 10⁶ food vacuoles per ml. of culture fluid. Since the bacteria contain 0·014 mg. NH₃ per 10⁹ bacteria, there was in the organisms 0·017 mg. exogenous NH₃ per ml. available for excretion. At the end of 12 hours the glaucomae had excreted 0·0182 mg. per ml. From these estimations it seems probable that a large part of the nitrogen available in the bacteria is eliminated as NH₃.

It is evident from the graphs that the rate of elimination of food vacuoles follows the rate of formation of ammonia and that the latter is apparently independent of the multiplication of the glaucomae. This is in accordance with Lawrie’s (1935) experience with Bodo caudatus. The cessation of ammonia elimination by Glaucoma after some hours of starvation also supports Lawrie’s suggestion that Bodo, when well-fed, metabolizes nitrogenous foodstuffs, but that under starving conditions in the presence of metabolic products the food requirements of the organism are met by preferential metabolism of non-nitrogenous food materials.

In the graphs, the curves for the elimination of food vacuoles are sigmoid. The apparent low rate of disappearance of food vacuoles during the first hour is probably due to the additional formation of some vacuoles after removal of the bulk of the bacteria. The low rate measured during the last 2 hours is due to the food vacuoles remaining in the slowly evacuating members of the population.

The curve for the decrease in volume in Exp. 20 is very irregular, owing to the rapidly changing nature of the population accompanying a high division rate. Consequently individual samples were inadequate. The values obtained were plotted and the statistically most probable line drawn.

The NH₃ curves are quite consistent up to their first peak, after which they are quite irregular. A sufficient number of experiments with multiple analyses for ammonia were carried out to convince us that the temporary disappearance of free ammonia from the solution is quite outside the limits of error of the method. We believe that the variations are quite real, but are unable to explain them.

We had hoped to be able to correlate the stage when ammonia is utilized from the medium with a change in respiratory quotient. Unfortunately, shaking in Warburg respirometers completely changes the time scale and we were unable to maintain parallel states of the organism between samples for ammonia and those for respiration. The following explanation of the disappearance of ammonia is tentatively put forward.

Pseudomonas furnishes Glaucoma with excess NH₃ which is excreted. It also furnishes other products which are stored as food reserves. When Glaucoma has used up all the available “metabolite A”, normal to this diet, it proceeds to the use of a similar but insufficiently aminated “metabolite B”, plus NH₃, from the medium. When “metabolite B “is exhausted and has been catabolized the NH, reappears in the medium where it remains at a fairly constant level, indicating a marked decrease or major shift in metabolism (possibly to endogenous sources, sensu stricto).

From the data of Exps. 19–21, and from earlier and subsequent observations, it seems most likely that the hypothetical curves drawn in Graph 4 most closely approximate the average conditions obtaining for experiments done under our conditions.

From these curves it is evident that the immediate source of nitrogenous waste material is the food organism, and that the nitrogen is eliminated from the glaucomae in the form of ammonia approximately 6 hours after the ingestion of bacteria.

The ciliate, Glaucoma, was cultured under conditions affording large populations uniform in relation to size, division rate, feeding rate and metabolic activity. The nitrogen entering the organism, the excretion of ammonia, the changes in size and number of cells and the rate of elimination of food vacuoles were measured and correlated.

It is concluded that most of the nitrogen in the bacterial food ingested by Glaucoma is eliminated in the form of ammonia approximately 6 hours after ingestion.