On the Hydrogen-Ion Concentration of Hay Infusions, With Special Reference to its Influence on the Protozoan Sequence

Abstract

It is well known that various animals in hay infusions appear and disappear in a regular sequence. With a view to ascertain how far the hydrogen-ion concentration of the medium is concerned in this matter, about 20 infusions made with hay and water from different sources have been kept under close observation, their pH and the state of some of the important organisms inhabiting them being examined periodically. The salt content of the medium has a considerable influence on both the hydrogen-ion concentration and on the metabolism of organisms; the culture fluids were titrated in order to determine the “alkali reserve* when the pH determinations were made. Peters (1906 and 1907) and Fine (1912) also examined the “phenolphthalein acidity” and “methyl-orange alkalinity,” or the “titratable acidity” of hay infusions, which, as they themselves recognise, is not a correct expression of the concentration of hydrogen ions with which the organisms are in contact. Moreover, they did not control the quality of the water which they employed for making the infusions. Further reference to their works will be made at a later stage in this paper.The infusions were prepared in the usual way—by mixing about 1 litre of water with 4 g. of dry hay in a glass jar; with this proportion of hay and water animals appear in 4 or 5 days, but if too much hay is used the medium becomes very acid and organisms do not appear for a long time, and sometimes not at all. The sources of the water employed were: (i) Cambridge tap water, pH 7·35 (corrected for salt error), alkalinity ·0042 N; (ii) Manchester tap water, pH 7·3, alkalinity ·0039 N; (iii) the Cam (river) water, pH 7·85, alkalinity 0039 N; (iv) distilled water, pH 7·30, practically free from alkalies. The hay was of a mixed variety, “meadow hay,” and was obtained from two different sources, the hay employed in infusions A-H being from a different place from that in infusions J-M. The quantity of water in the infusions varied from 1 litre to 15 litres, but the proportion of hay was the same in all. The mouths of the jars used for the infusions were very wide, and they were kept uncovered so that the air might have full effect. Naturally there was a’ great loss of water due to evaporation and in the case of small cultures, fresh water from the original source had to be added to keep them going; a careful account of the water added was kept. As controls, in one infusion distilled water was periodically added to make up for the loss due to evaporation and in another the influence of the atmosphere was excluded by covering the infusion with a thick layer of paraffin oil.The infusions were kept in a well lighted room at two different places, at a distance of 1 ft. and 12 ft. respectively from windows facing north. The temperature of the room during the period of the experiments varied between 13–17° C.All the infusions were “unseeded,” i.e. they were not inoculated with any organism when they were set up.The pH determinations were made by colorimetric methods, using the sulphon-phthalein indicators of Clark and the Söensen’s phosphate and Palitzsch’s borax-boric acid solutions as buffer mixtures. Corrections for the salt error were made using Saunders’ curve (1923).The titrations for the alkali reserve were made with ·01 N H2SO4. Methyl-orange could not be used with convenience as an indicator because the culture fluid becomes turbid and brown within a few days of starting the infusions, so ·04 per cent, brom cresol purple was used instead. This indicator, as is well known, is affected by CO2; the liquid had therefore to be boiled to expel this gas. The end-point was assumed to have been reached when the liquid remained yellow permanently after boiling for 2 or 3 minutes.The hay was always kept uniformly distributed in the culture, so that both the pH and the alkalinity of the liquids taken from different places and depths of an infusion were the same; the greatest number of organisms was always found just below the surface, and all the measurements recorded in the following experiments were of samples taken from near the surface of the infusions. Woodruff (1912) observed that a definite sequence of forms is not apparent at the middle or bottom of the infusions.The infusions under observation were designated A, B, C…M (see Table 1, cols, 1, 2 and 3). The course of a single experiment (infusion E), which may be regarded as typical, is shown graphically in Fig. 1. The results can be briefly summarised as follows :The range of the hydrogen-ion concentration can be conveniently divided into three phases. In the first, just after the infusions are set, the pH suddenly goes down, i.e. the acidity rises. Sometimes, however, as in infusions E and F, before the sharp fall began, the pH rose for the first few days, for 3 days in E and for 1 day in F, reaching 7·7 and 7·65 respectively; in infusion D, the pH remained stationary for the first day. The maximum acidity in infusions made with Cambridge tap water, e.g. C-F, is reached within 3 or 4 days after the beginning of the fall when the pH goes down to 6·5–6·6. In infusions made with distilled or Manchester tap water, G-K, the pH goes on falling for 7 or 8 days, being sometimes as low as 5·5 (J) at the end of that period. Obviously the lack of alkali reserve in these waters allows the pH to reach such a low level. In infusion M, made with the river water (pH 7·85), the acidity did not go higher than pH 7·05 ; this was reached in 2 days and remained at that point for the next 10 days. In col. 5 of Table I the age of the different infusions when their pH was minimum is given.In the second phase, the pH begins to rise rapidly, the rate of rise, as was the case with the rate of fall, depending upon the nature of the water used and on the size of the infusion. For example, amongst the Cambridge tap water series, A-F, in A and B, which were six times bigger than infusions C-F, pH 7·35 (the pH of Cambridge tap water, corrected for salt error) was reached at the age of 25–34 days, while in C-F, the same point was reached when the infusions were only 16 days old. The rate of rise in the distilled and Manchester water infusions is much more rapid than in in the Cambridge tap water cultures, e.g. while in infusion C-F it took 13, 6, 14, 10 days respectively for the pH to become 7·35, in infusions G and H, not-withstanding the fact that the pH in them had fallen much lower, it took only 6 days. In infusions J and K, however, also prepared from distilled and Manchester waters, but with hay from a different source, at first the rise in pH was as rapid as in G and H,pH 6·5 and 6·85 being reached in 2 and 5 days respectively, but later on it was much slower, pH 7·35 being reached in 43–45 days. In the river water infusion, M, there was a sudden jump from the lowest pH reached, 7·05, to 7·35 on the twelfth day, and it remained at that level for the whole of the next week. In col. 6 of Table I is given the age when the pH in the various infusions reached 7·35.In the third phase, the pH rises slowly and its curve is consequently almost straight with an upward tendency. The relative rate of rise, as far as the size of the infusions is concerned, is the same as in the second phase, i.e. it is slower in larger cultures, but with regard to the quality of water it is opposite, i.e. in the distilled and Manchester water infusions it is slower than in the Cambridge tap water ones instead of being more rapid as it was in the second phase (see col. 9 of Table I).The maximum pH reached in the infusion was about 8·7, no further rise being noticed though the infusions were kept under observation for 2 months after this point had been reached.On the whole, the curve showing the increase in alkali reserve follows almost the same course as that of the pH. In the first phase when the pH is falling, the alkali reserve also decreases in amount; for instance, in the Cambridge tap water infusions, it falls from ·0042 N to as low as ·0030 N(infusion F). In the Manchester and distilled water infusions, which start with very little alkali reserve, this decrease is also visible if the pH falls very much, e.g. infusions J and K.In the second phase, when the pH rises rapidly, while in the Cambridge tap water infusions, e.g. E and F, the concentration of the alkalies also rapidly increases, in the distilled and Manchester water infusions, G-K, it does so gradually. The rate of increase is more rapid in smaller infusions than in larger ones (cf. C-F with A-B). This seems to be due to evaporation. But at a particular pH, say 7·35, the water with which the infusions are made remaining the same, the alkali reserve in the various infusions is almost the same, that in the larger infusions being only slightly less (col. 8 of Table I). In this connection it is worth while emphasising that the difference between the times required by the large and small infusions to reach the above pH and concentration of alkali reserve was relatively great; this means that the pH and alkali reserve of an infusion are affected by one another more closely than by the age of the infusion.In the third phase when the pH rises gradually, the carbonates also increase in concentration, but a relatively great change in the concentration is accompanied by only a small change in the pH, i.e. the parallelism between the two curves is not very close. This also probably accounts for the fact that at a higher pH, say 8 · 0, unlike that at pH 7 · 35, the alkali reserve in different infusions made with the same quality of water is different (col. 10 of Table I).In the case of infusion E the pressure of CO2 was also calculated, using the following formula after Saunders (1926): The curve (Fig. 1) shows the maximum pressure of CO2 (over 26 mm.) on the seventh day when both the pH and the alkali reserve were minimum. After the twentieth day the CO2-pressure showed a rapid decrease, being only 7 mm. on the thirtieth day, then it fell slowly, being about 4 mm. on the ninetieth day.Before going further, it seems necessary to make some general remarks on the hydrogen-ion concentration, and the alkali reserve. As will be mentioned in the biological section, during the first few days when an infusion is set, there are no organisms except the bacteria, which are in great abundance. The bacteria apparently cause the fermentation of the organic material of hay, producing CO2 which makes the infusion acid with the consequence that the pH falls quickly. The greater amount of acidity in the distilled and Manchester water infusions seems to be due to the absence of sufficient alkali reserve to neutralise the CO2 which is being produced. The decrease in the concentration of alkali reserve in this period which is specially noticeable in the Cambridge tap water infusions can be explained as being due to the dissolving action of acids higher than carbonic acid, e.g. acetic, formic, butyric, etc., which, as is well known, are prodúced in the course of bacterial growth. The tables of Reddie (1923) also show a decrease in the alkali during the first few days. But there seems to be some other factor also involved, because when tap water was allowed to stand in a wide-mouthed bottle in which no hay was added, the concentration of alkalies decreased just as it does in normal hay infusions, though the rate of decrease was much slower. Starting with the concentration ·0042 N, it was ·0034 N after 7 days (in infusions this figure is reached in 3 days) ; ·0030 N after 15 days, and ·0020 N after 35 days, reaching ·0010 N at the end of 125 days (Table II). The pH of the standing water rose, being 7 ·5 at the end of 35 days; this is probably due to the diffusion out of CO2. It appears that the size of the mouth of the vessel has an important bearing on this question, because in another bottle which had a very narrow mouth, the standing tap water decreased to ·0039 N only in the course of one month. Also in another similarly narrow-mouthed bottle in which tap water was standing since March, 1925, the concentration of alkali reserve measured on January 26th was ·0010 N. The fact that the rate of decrease is more rapid in wide-mouthed vessels suggests that exposure to air is intimately concerned in this matter.The increase in the alkali concentration in the second and third phases, is of course due to the progressive mineralisation of the organic matter (cf. Reddie). With the increase of alkali reserve, the pH naturally rises.Atmosphere seems to have a great influence on the course of development of hay infusions. To see its effect one infusion, immediately after it was set, was covered with a thick layer of pure paraffin oil. Within a few days, as in normal infusions, the pH went down, but it did so considerably, reaching 5 ·0. Moreover, it never rose above this point till the oil was removed after 35 days. One reason for this phenomenon seems to be that the oil did not allow the CO2 to diffuse away. Of course, during the period that the pH was about 5 ·0 no protozoan appeared in the infusion. The scum which, as will be mentioned in the next section, sets on the surface of a normal infusion within 3 or 4 days, appeared in very small amount in this infusion. After 2 months, when the oil was removed the pH began to rise, animals appeared and the infusion ran its natural course.Before describing the sequence of animals, a few remarks may be made on the gerneral course of the development of hay infusions. Within 48 hours after an infusion is set, the water becomes pale yellow, remaining, however, quite clear. In the next 2 or 3 days the water becomes turbid and a bacterial scum forms on the surface of the infusion. The period during which these changes take place corresponds to the first phase of pH range, during which the pH is falling sharply. During this time no organism except bacteria is found in the cultures. The scum appears about the time when the pH just begins to rise, i.e. the beginning of the second phase. The scum has an important bearing on the biology of the infusions, especially in their early history, as, until this sets in, no animal makes its appearance in the cultures. As the infusion progresses, the scum becomes darker and opaque and at about pH 7 ·7 it no longer floats on the surface but settles down on the hay. By this time the general colour of the infusions is brown or dark brown. Algae generally appear at about pH 7 ·8. Of the infusions under observation algae appeared in A and B only, the big cultures. With the appearance of these organisms the coloration of the infusions turns greenish and ultimately dark ; the latter change seems to be due to the presence of fungi.As is well known, a great variety of organisms appears in hay infusions*. But for the purpose of the problem under investigation a close study of a few that occur regularly is quite enough ; so during the course of these experiments attention was paid to the following organisms only: Holophyra, Plagiopyla, Colpidium, Para mecium, Amphileptus, Monas, GastrostylaHolophyra. This is generally the first animal to be found in the infusions, especially in those that are made with Cambridge tap water. It appears just on the day when the scum sets in and it remains in the cultures for about 10 days, multiplying by repeated binary fission during this period. Its pH range is 6 ·5 –7 ·4. In the distilled and Manchester water infusions (G and H), which were made with hay from the same source as the Cambridge tap water infusions, Holophyra did not appear at all, while in infusions J and K, made with hay from a different source from the above, it appeared but did not flourish well and died off in 5 days. In the river water infusion, M, made with hay from the same source as J and K, Holophyra flourished much better. As was mentioned in the previous section, distilled and Manchester water infusions become highly acid and remain so for a considerable time; it therefore seems as though Holophyra cannot stand a high acidity.Plagiopyla. This has generally the same history as Holophyra; both flourish together in infusions. It appears about 24 hours later than that animal and lasts about 12 days. Its pH range may be said to be 6 ·6 –7 ·5. Like Holophyra, it did not appear at all in infusions G and H and was found in small numbers only in J’ and K.Colpidium is one of the most important organisms of hay infusions. It flourishes in both the Cambridge tap water and distilled water infusions. In the Cambridge tap water cultures it appears after Holophyra and Plagiopyla, at pH 7 ·0 –7 ·2, but in the distilled water infusions, where it is the first organism to appear, it is found at pH 6 ·0 –6 ·5, and sometimes even at a lower pH. This means that Colpidium can stand a very high acidity. Peters also observed a similar state of things. As regards the upper pH limit, Colpidium can persist till pH 8 ·5. Its actual condition in an infusion depends upon the activity of the next animals in the sequence, which are generally Paramecium. For example, in infusion A, between pH 7 ·7 –8 ·0 when Paramecium were in their vigour, Colpidium declined in numbers, but after pH 8 ·0 they again became prominent. In infusions E and F, Paramecium were flourishing till 8 ·45. Colpidium, which were in small numbers till that pH, became very common subsequently; in infusion C in which Paramecium never predominated, Colpidium flourished uniformly till pH 8 ·5.In infusions J and K, Colpidium almost disappeared at a pH about 7 ·0, this was probably due to the appearance of Amphileptus which, as will be described below, flourishes alone in infusions.Beyond pH 8 ·2 Colpidium become very much swollen and show many vacuoles in their endoplasm. Spirostomum also swells at a high pH (Saunders, 1924).As was mentioned in the introductory section, water had to be periodically added to most of the small-sized infusions to keep them going. Whenever this was done, and Colpidium were present, they were very adversely affected. On the other hand, it appeared that Paramecium were indifferent or affected in an opposite direction.Paramecium. It seems curious that Woodruff did not obtain Paramecium in any of his unseeded cultures when set in a similar way to mine. It has been the practice in this laboratory to produce Paramecium for class work simply by adding hay to tap water. The method has never been known to fail.Paramecium was found in infusions A-J. In the larger cultures, A and B, it appeared at pH 7 ·0 –7 ·2, while in the rest, which were much smaller, it did so at pH 7 ·5 –7 ·7-It may be mentioned that the various infusions did not reach this pH (7 ·5 –7 ·7) at the same age, there was a considerable difference; for example, while C-F were 20 –30 days’ old at this pH, G-J were 60 –70 days of age. This clearly shows that the appearance of Paramecium depends upon the pH, rather than upon the age of an infusion.From what was said in the case of Colpidium, it follows that the numbers of Paramecium at a particular time depend upon the activity of that animal ; either one or the other predominates at a time, but Paramecium may be said to be at the height of its vigour between pH 7 ·8 –8 ·0. Saunders also reached the same conclusion. But as regards the upper pH limit, while Saunders did not find Paramecium in cultures having a pH higher than 8 ·0, in my infusions Paramecium occurred even at pH 8 ·5. It appears, however, that the company of Rotifers is not congenial to Paramecium, as in infusions in which Rotifers became prominent (e.g. A) Paramecium disappeared at pH 8 ·0.Unlike Colpidium, Paramecium cannot stand a low pH. Some Colpidium and Paramecium from an infusion at pH 7 ·8 were put in a watch-glass to which was added culture fluid from another infusion which had pH 6 ·0. Within half an hour all the Paramecium died, while the Colpidium were not affected at all.Saunders found that Paramecium show a decided preference for their own culture fluid to tap water having the same pH (raised by shaking the water, etc.). He did not hazard any explanation. I put an equal number of Paramecium and Colpidium into two watch-glasses, having tap water and culture fluid (of same pH) respectively and found that those in the culture fluid reproduced and flourished much better than those in the tap water. This may have been due to the greater amount of carbonates in the infusion water which in this experiment was .0060 N while in tap water it was .0042 N.In infusions J, K, M, made with hay from a different source from the rest, the Paramecium which occurred appeared to be a different variety from those in cultures A-H. They flourished in the same pH range as the other Paramecium.Amphileptus occurred in infusions J, K and M (made with hay from a different source from the rest). Though it remains in infusions for a short period, about a week, its effect on other animals in the sequence is quite profound. For example, Colpidium, which otherwise is quite a hardy animal, disappears at once when Amphileptus comes into prominence. As regards the pH range, Amphileptus was found between pH 6 ·8 –7 ·1 ‘n infusions J and K and between pH 7 ·3 –7 ·5 in infusion M. It was followed by Monas.Gastrostyla is an animal that does not seem to be influenced at all by a high or low pH. It was found, though never in large numbers, at all times and in all the infusions.It is evident that most of the organisms in hay infusions have a definite pH range. For example, Holophyra and Plagiopyla do not persist beyond pH 7 ·5, Paramecium does not appear till the pH is about 7 ·0. The pH range of an organism is liable, however, to be disturbed by the adverse influence of another organism in the infusion, e.g. effect of presence of Amphileptus on Colpidium, etc.The actual organisms that appear in an infusion depend upon the quality of water and hay used for making the infusion. But the sequence of appearance is always the same, the change consisting merely of the omission of one or other organism. In infusions such as were used in my experiments this sequence was : Holophyra, Plagiopyla, Colpidium, Amphileptus, Paramecium.I take this opportunity of expressing my thanks to Mr J. T. Saunders, M.A., who made valuable suggestions and criticisms during the investigation.