Distribution of Soil Protozoa.

For both the bacteria and algæ observations have been made regarding their distribution through successive depths of the soil; little can be said, however, about the protozoa in this connection. It is certain that they occur throughout the first six inches of the Rothamsted soils, though their relative frequencies in the successive inches has not been determined, but probably they are most abundant in the 2nd to the 4th inch.

In this country experiments have not been made to determine whether sub-soil normally contains protozoa; but from some South African soil, taken under sterile conditions 4 ft. down and examined in this laboratory, large numbers of protozoa were cultivated.

This soil, however, could not, for various reasons, be regarded as a typical sub-soil.

Kofoid records the presence of Nægleria gruberi in clay and rock talus taken from the sides of excavations of over 20 ft. depth, but the possibility of external infection does not appear to have been excluded.

The presence of protozoa is not peculiar to British soil since they have been found by various workers in Germany, France, the United States, and elsewhere. In view of their probable importance in the soil economy there has been instituted a survey of the protozoan species of soil from all parts of the world.

This work is in charge of Mr. Sandon, to whom I am indebted for the following summary of his as yet unpublished research.

“The majority of soil protozoa (like the fresh-water forms) appear to be quite cosmopolitan, for the species found in such widely separated localities as England, Spitsbergen, Africa, West Indies, Gough Island (in the South Atlantic) and Nauru (in the Pacific) are, with few exceptions, identical. This distribution indicates an ability to withstand an extremely wide range of conditions, for the same species occurring in Arctic soils, which are frozen for the greater part of the year, are found also in soils exposed to the direct rays of the tropical sun. Even sand from the Egyptian desert contains protozoa, though it seems probable that in such cases they must be present only in the encysted condition for the greater part of the time.

“Not every sample of soil, however, contains all the species capable of living in soil, but the local conditions determining the presence or absence of any species are at present unknown. In general the numbers, both of species and of individuals present, follow the number of bacteria. They are consequently most numerous in rich moist soils. The statement sometimes made that protozoa are most numerous in peaty soils is based solely on the number of Rhizopod shells found in such localities; but as most of these shells are empty, their abundance is probably due simply to the slowness with which they disintegrate in these soils where bacterial activity is low, they do not indicate a great protozoal activity. Active protozoa do occur even in extremely acid soils, but their numbers in such cases are low. The common soil protozoa, in fact, appear to be as tolerant of differences in soil acidity as they are of differences in climate, for many of the same forms which occur in acid soils are found also in soils containing high percentages of chalk. It is possible that some of the less common species may be confined within closer limits of external conditions but the information available on this point is inadequate. All the species, however, which in Rothamsted soils occur in the highest numbers (e.g. Oicomonas termo, Heteromita spp., Cercomonas crassicauda, Nægleria gruberi, Colpoda cucullus, C. steinii) occur in practically every soil which is capable of supporting vegetation, though, of course, in very varying numbers.”

It is evident, therefore, that the protozoa must be regarded as constituting part of the normal micro-organic population of soils, and as such are probably playing an important rôle. Unfortunately our knowledge of the physiology of these organisms is extremely scant, and much of future research must be directed towards elucidating their functions and their responses to varying environmental conditions.

CHAPTER V.
PROTOZOA OF THE SOIL, II.

In the preceding [chapter] an outline has been given of the development of the study of soil protozoa, with especial reference to its qualitative aspects.

Here it is proposed to deal with the quantitative methods which have been devised for studying these organisms and the results obtained.

From the beginning great difficulty has been encountered in finding means for counting protozoa; and most of the early results have been obtained by the use of one of the following methods: (1) direct counts in a known volume of soil suspension by means of a microscope; (2) dilution method as used for counting bacteria, and suggested by Rahn, who made dilutions of the soil and determined, by examination at periodic intervals, the one above which protozoa did not grow; (3) Agar plating as used by Killer; (4) counting per standard loop of suspension as devised by Müller. Of these the two last have been little used, and for various reasons are now discarded by most workers. Direct methods have been used extensively in the United States by Koch[13] and others,[16] who claim to have got satisfactory results; they are, however, highly inaccurate and should be discontinued. The present writer[3] has shown that there exists a surface energy relationship between the soil particles and the protozoa, so that the two are always in intimate contact; thus rendering it impossible to count under the microscope the number of organisms in a given weight of soil suspension ([Fig. 9]). Further, in a clay soil, such as is found at Rothamsted, the clay particles alone make it very difficult to use such methods.

The demonstration of this surface energy relationship affords an effective rejoinder to the criticism made against Russell and Hutchinson’s hypothesis, viz., that soil protozoa must be very few in numbers, since it was impossible to see them on examining soil under the microscope.

Fig. 9.—Showing the number of amœbæ and flagellates withdrawn from suspensions of varying strengths by different types of solid matter. A = clay: B = partially sterilized soil: C = ignited soil: D = fine sand: E = waste sand. Since complete withdrawal occurs when the numbers of organisms added are less than the capacity of the solid matter, the first part of each of the above curves is coincident with the ordinate. The numbers of organisms are given in thousands. (From Journ. Agric. Soc., vol. ix.)

X-axis: Number of Organisms per c.c. left in Solution.

Y-axis: Number of Organisms per c.c. taken up by Solid Matter.

The second or dilution method is the one, therefore, that has been most extensively developed.

Cunningham obtained concordant results in this way, and his method, modified by L. M. Crump, was as follows: 10 grams of soil were added to 125 c.c. of sterile tap-water and shaken for three minutes. This gives a 1 in 12·5 dilution. From it further dilutions were made until a sufficiently high one was obtained. Petri dishes, containing nutrient agar, were inoculated with 1 c.c. of each of the dilutions and incubated. At intervals covering 28 days the plates were examined and the presence or absence of protozoa on each recorded. In this way the approximate number of organisms per gram of soil could be found.

By methods essentially similar to this numerous counts have been made of the bacteria and protozoa in field soil and in partially sterilized soils. They were, however, inconclusive; thus, on the one hand, Goodey and several American observers, found no correlation between the numbers of protozoa and bacteria, while Miss Crump and Cunningham obtained evidence pointing to the reverse conclusion.

Such divergence of opinion was probably mainly due to two causes: firstly, that the time elapsing between the successive counts was too long, for it has been shown recently that the number of bacteria and protozoa fluctuate very rapidly; and secondly, the method was not completely satisfactory since only the total numbers of protozoa were considered, no means having been found of differentiating between the cystic and active forms. This was a particularly serious source of error for it is possible for soil to contain large numbers of bacteria and protozoa, of which a high percentage of the latter are in the form of cysts. A count made on such a soil would give results apparently opposed to the theory that protozoa act as depressors of bacteria.

This difficulty has, however, been overcome by a further modification of the dilution method, and it is now possible in any soil sample to count both the numbers of cysts and active forms. Also a further advance in technique has made it possible to recognise and enumerate the common species of protozoa, instead of simply grouping them as Ciliates, Flagellates, and Amœbæ, as was done in the past.[7]

Briefly the method consists in dividing the soil sample into equal portions (usually 10 grams each) one of which is counted, thus giving the total numbers of protozoa (active + cystic) present. The second portion is treated over-night with 2 per cent. hydrochloric acid, the HCl used being B.P. pure 31·8 per cent. Previous experiments have shown that such acid kills all the active protozoa, leaving viable the cysts. The number of cysts is therefore found by counting this treated sample, and the number obtained subtracted from the total gives the active number.[F]

[F] The proof of the accuracy of this method will be found in the following papers:—

(1) Cutler, D. W. (1920), Journ. Agric. Sci., vol. x., 136-143.

(2) Cutler, D. W., and Crump, L. M. (1920), Ann. App. Biol., vol. vii., 11-24.

The discovery of this method at once puts into the hands of the investigator a much more efficient instrument for studying the activities of the soil micro-population, especially since at a slightly later date Thornton’s method for counting bacteria was devised.

Early in 1920 Cutler and Crump[6] decided to make a preliminary survey of the protozoon and bacterial populations of one of the Rothamsted field soils (Broadbalk dunged plot). The investigation was continued for 28 days, daily soil samples being taken. The results so obtained showed that an extended investigation of the micro-population of field soil would yield interesting and important results, especially as it was evident that certain views held by soil biologists required modification.

In July of the same year, therefore, it was decided to start an extended investigation of the soil protozoa and bacteria. The method adopted was to make counts of the numbers of bacteria and of six[G] species of protozoa in soil samples taken daily direct from the field (Barnfield dunged plot) and by statistical methods to correlate these counts one with another and with the data for external conditions. Observations at shorter periods than 24 hours could not be made, but it was found possible to continue the research for 365 days.[7]

[G] Actual counts were made of six species, though, as stated on [p. 10], observations were made on seventeen.

Fig. 10.—Daily numbers of active amœbæ (Dimastigamœba and Species α) and bacteria in 1 gram of field soil, from August 29 to October 8, 1920. (From Phil. Trans. Roy. Soc., vol. ccxi.)

X-axis: August September October

Y-axis (left): Amoebae Active numbers per gramme of soil

Y-axis (right): Bacteria in millions per gramme of soil

Legend: Dimastigamoeba
Species α
Bacteria

The number of all the organisms showed large fluctuations of two kinds, daily and seasonal. The size of the changes that took place within so short a period as 24 hours was, perhaps, the most surprising fact that the experiment revealed. Thus three consecutive samples gave 58·0, 14·25 and 26·25 millions of bacteria per gram respectively; and the changes exhibited by any of the species of protozoa were at times even larger. This fact is of extreme importance, since in the past it has always been assumed that the number of bacteria remained fairly constant from day to day, and investigators have not hesitated to separate the taking of soil samples by long periods. It is now obvious that such a procedure is of little use for comparative purposes ([Fig. 10]).

It has usually been assumed that the changes in the external conditions markedly affect the density of the soil population. To test this the environmental conditions—temperature, moisture content and rainfall were examined; but contrary to all expectation no connection could be traced between any of these and the daily changes in numbers of any of the organisms investigated, and moreover the species of protozoa appeared in the main to be living independently of one another.

It is difficult to believe that external conditions are as inoperative as appears from the above; and in view of the known complexity of the soil it is possible that further research will show that certain combinations of external conditions are important agents in effecting the changes.

Fig. 11.—Numbers of active amœbæ (Dimastigamœba and Species α) and bacteria to 1 gram of field soil for typical periods in February and April, 1921. (From Phil. Trans. Roy. Soc., vol. ccxi.)

X-axis: Feby. Feby. April

Y-axis (left): Amoebae Active numbers per gramme of soil

Y-axis (right): Bacteria millions

Legend: Dimastigamoeba
Species α
Bacteria

In the case of the bacteria, however, the agent causing the fluctuations is mainly the active amœbæ. This was well shown during the year’s count, for with only 14 per cent. of exceptions, 10 per cent. of which can be explained as due to rapid excystation or encystation, a definite inverse relationship was established between the active numbers of amœbæ and the number of bacteria ([Figs. 11] and [12]). Thus a rise from one day to the next in the amœbic population was correlated with a fall in the numbers of bacteria and vice versa. It must not be supposed that the flagellates are of no account in this process; some species, known to eat bacteria, undoubtedly induce slight depressions, but, owing to their small size, any effect is masked by the greater one of the amœbæ.

Fig. 12.—Numbers of active amœbæ (Dimastigamœba and Species α) and bacteria in 1 gram of field soil for typical periods in September, October, and November, 1920.

X-axis: August September October

Y-axis (left): Amoebae, thousands

Y-axis (right): Millions, Bacteria

Legend: Dimastigamoeba
Species α
Bacteria

These experiments seem to admit of no doubt that in field soil the active protozoa are instrumental in keeping down, below the level they might otherwise have attained, the numbers of bacteria; but a further proof of this contention ought to be obtained by inoculation experiments. It should be possible, by inoculating sterile soil with bacteria alone and with bacteria plus protozoa, to demonstrate fluctuations in bacterial numbers in the latter, while those of the former remained constant. This admittedly crucial test has often been tried, but owing to difficulties in technique, etc., has always failed. Recently, however, by using new methods confirmatory results have been obtained.[5]

Ordinary field soil was sterilised by heat at 100° C. for 1 hour on four successive days; it was then divided into equal portions, one of which was inoculated with three known species of bacteria, and the other inoculated with the same number of bacteria plus the cysts of the common soil amœba Nægleria gruberi. The numbers of bacteria in each soil were counted daily for the first eight days and then daily from the 15th to the 21st day after the experiment started. The results are given in [Table VII.] and [Fig. 13].

TABLE VII.

Numbers
of Days
after
Inoculation.		Control
(Bacteria
alone).		Control
Bacteria
+ Amœbæ.
 0 13·0 12·2
 1 48·6 35·4
 2 97·6117·2
 3127·0178·4
 4154·8154·4
 5196·8177·0
 6214·4151·8
 7193·4 75·6
 8165·2 65·8
15169·2 72·8
16174·8 30·2
17175·6 53·2
18168·4 82·8
19160·4 43·8
20171·2 70·8
21176·2 28·2
The numbers of bacteria are given in
millions per gram of soil.

Fig. 13.—Numbers of bacteria counted daily in soils containing

A. Bacteria alone.
B. Same Bacteria as in A + Amœbæ.
C. Same Bacteria as in A + Flagellates.

(From Ann. Appl. Biol., vol. x.)

It will be noted that the numbers of bacteria in each soil rose steadily until a maximum was reached 6-8 days after inoculation. This is in accordance with expectation, since the reproductive rate of bacteria is much greater than that of the amœbæ, which, until their active forms are numerous, will not exert any appreciable influence on the bacterial population. Further, since the protozoa were inoculated as cysts an appreciable time would elapse before excystation took place. The last seven days of the experiment are of particular interest. During this period the amœbæ were known to be active in the soil, and were depressing the bacterial numbers, for in the control (protozoa-free) soil the variation in numbers was within experimental error, while in the other soil the variations were considerable and well outside experimental error. In fact the variations were comparable with those found from day to day in untreated field soils. Finally, the experiment shows that the bacteria in protozoa-free soil are able to maintain high numbers for a longer period than those living in association with protozoa.