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Full text of "Some Notes on Soil Protozoa"

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III. Some Notes on Soil Protozoa. 

By C. H. Martin, M.A. y and K. R Lewin, B.A. , Rothamsted Experimental 

Station, 

Communicated by Horace T. Brown, F.Ii.S. 

(Received December 5, 1913, — Read February 5, 1914.) 

[Plates 5 and 6.] 
Contents. 

PAGE 

I. Introduction 77 

II. Methods of Preparation and Culture 80 

III. Cucumber bed — Vahlhampfia soli, n. sp., Ammba cucumis, n. sp 82 

IV. Seedling bed — Amoeba gobanniensis, n. sp 89 

V. Conclusions 91 

VI. Literature List 92 

VII. Description of Plates : 93 

Introduction. 

It is rather a curious fact that it has been left to agricultural chemists to bring 
into prominence the important part that free-living Protozoa may play in the soil. 
At the present time it seems to us that the prevalent idea as regards the distribution 
of free-living protozoa is that they can exist in the trophic state only in definite 
accumulations of fresh water (e.g., pools, rivers, lakes) or in the sea. It seems also to 
be generally held that after the drying up of small pools the cysts of protozoa are 
transported by currents of air, and that it is to these wind-borne cysts that the 
prevalence of protozoa in cultures of ordinary soils may be mostly attributed. It is 
very difficult to trace the historical origin of this view, but it is evident that to the 
early workers on protozoa the idea of this limitation of their active life to water was 
unthought of. 

It is unnecessary to refer here to the works of very early writers, by whom the 
spread of disease was sometimes attributed to invisible forms of life present in the air. 
It would seem, however, from the following quotation that Ehrenberg held a 
far wider view as regards the distribution of protozoa than that which is fashionable 
at present. In his great work ' Die Infusionsthierchen als Vollkommene Organismen/ 
p. 496, he says :— 

"Jeder der bekannten lebenden Korper besitzt eine Organisations-Feu chtigkeit. 
So lange er diese, den ihn bestiirmenden physikalischen Naturkraften entgegen- 
kampfend, in seinen Hauptorganen erhalt, so lange ist er lebend ; sobald sie durch 

(315.) Published separately, May 30, 1914. 



78 MESSRS. C. H. MARTIN AND K. R. LEWIN : SOME NOTES ON SOIL PROTOZOA. 

Hitze Frost oder eigene Schwache verloren geht, oder durch und durch erstarrt, 
erfolgt der Tod, der auch auf nianche andere Weise eintreten kann. Diese Organisa- 
tions-Feuchtigkeit nehmen Kaferlarven im dtirrsten Holze, Mottenlarven im diirrsten 
Pelze, Infusorien und Mooswurzeln, Samen, dergl., im diirrsten Sande aus dem 
Dunste der Atmosphare in sich auf, bleiben fleischig und feucht und nassen sogar 
ihre Umgebung. Lebende Dammerde bleibt feucht/ 7 

It is important to note that at the time this work was written the cysts of Protozoa 
were still unknown, and that therefore, for Ehrenberg, life meant trophic life, and 
not latent life in the cyst. 

By later workers this wide view of the distribution of protozoa seems to have 
become gradually more limited. For example, Dujardin in his account of the 
protozoa in the ' Histoire Naturelle des Zoophytes/ 1841, p. 169, states : — 

" Certains Infusoires vivent, non pas simplement dans les eaux, mais dans les sites 
habituellement humectes, comme les touffes de mousses, et surtout les couches minces 
d'oscillaires, sur la terre ou sur les murs humides ; pour les trouver, il suffit d'agiter 
et de presser dans un vase d'eau successivement plusieurs touffes de mousse prise au 
pied des arbres, dans les lieux frais, ou au bord des ruisseaux ; ou bien de placer dans 
une soucoupe, avec un peu d'eau, la pellicule enlevee a la surface du sol couvert 
d'oscillaires." 

From this quotation it is evident that Dujardin regarded the existence of the 
Protozoa in their trophic state in some saturated soils as certain, but even in the case 
of saturated soils later writers seem to have believed that the forms met under these 
conditions were mostly present as cysts. It is to Stein that we owe the discovery 
that nearly all free-living protozoa can encyst, and it is probably to him also that we 
owe the present limitation of our view as to the distribution of protozoa. On p. 20 
of his work ' Der Organismus der Flagellaten' (1878) he states : — 

" Die encystirten Infusionsthiere erwachen, wenn sie fruher oder spater unter 
Wasser gesetzt werden, zu neuem Leben und durchbrechen meist schon nach wenigen 
Stunden ihre sich erweichende und aufquellende Cyste. Sie konnen aber auch in 
trockenem Zustande, gleich Pflanzensamen und Pflanzensporen, durch die Winde auf 
weite Entfernungen hin fortgefuhrt und auf den verschiedensten Gegenstanden, z. B. 
auf dem ausgebreiteten Heu der Wiesen, auf den Moosliberzligen alter Gemauer, 
zwischen der rissigen Rinde alter Baume oder im Sande der Dacher abgesetzt werden. 
Uebergiesst man dann solche Gegenstande mit Wasser, so liefern die daran haftenden 
Cysten ebenfalls schon nach wenigen Stunden die freie bewegliche Infusorienform." 

This view seems to have appealed to Butschli since in his work on Protozoa 
(I Abtheilung, Sarcodina und Sporozoa, 1880-82) he states on p. 162 : — 

" So treffen wir Amoben in feuchtem Sand oder Moos von Baumen, und zwar sowohl 
am Fusse solcher als in ziemlicher Hohe tiber dem Erdboden an ... , 



MESSRS. 0. H. MAETIN AND K. ft. LEWIN: SOME NOTES ON SOIL PKOTOZOA. 79 

" Dass es sich in diesen Fallen meist urn Formen handelt, die durch Winde im 
encystirten oder zum Theil vielleicht auch nicht encystirten Zustand gewissermaassen 
verschlagen wurden, dilrfte keinem Zweifel unterliegen. 

"In dieselbe Kategorie diirfen wir vielleicht auch die von Cienkowsky auf 
Pferdemist beobachtete Diplophrys stercoreum und das unter ahnlichen Y erhaltnissen 
getroffene Platoum stercoreum, sowie den jedenfalls zur gleichen Gattung gehorigen, 
von Gabriel in feuchter, mit thierischen Excrementen durchsetzter Erde gefundenen 
sogen. Troglodytes rechnen, deren nachste Verwandte ja das slisse Wasser 
bewohnen." 

Finally it is to this view of the distribution of protozoa that we probably owe a 
recent interesting paper by E. M. Ptjsohkakew.^ Puschkarew left sterilised plates 
exposed to the air and examined the cultures that he so obtained. Of the 13 forms 
he obtained in this way all can be found in soil, but it seems fairly clear that he 
believed that nearly all these forms in his cultures owed their origin to cysts from dried 
pools on the banks of the river which had been transported by the wind, cp. p. 331. 

" Herbst und Winter 1910/11 waren nun zum Teil sehr regnerisch und also 
ungiinstig fur meine Arbeit. Nun kam aber das Fruhjahr 1911 mit relativ wenig 
Regen und endlich der Sommer mit seiner grossen Hitze ; viele Siimpfe waren ganz 
ausgetrocknet ; das Wasser in Fliissen und Seen stand kolossal niedrig ; der meistens 
mit Algen bewachsene Fluss- und Seeboden war zum Teil von Wasser befreit ; die 
ausgetrockneten Algen waren mit Protozoencysten bedeckt und die Luftstromungen 
konnten letztere, zusammen mit feinen Schlammteilchen, vom Boden in die Hohe 
reissen und zu entfernten Orten transportieren." 

Of recent years a large amount of our knowledge of that difficult group the Amoebsa 
has been obtained from animals cultivated from soils (Nagler and Glaser), but it is 
interesting to note that little if any attention has been paid to the biology of the 
forms thus obtained, in their natural state ; and we believe that to most biologists the 
idea that ordinary soils contain a large number of protozoa in an active condition is a 
new one. The view that the presence of such protozoa in excessive numbers may be 
the cause of soil sickness was, we believe, first put forward by Russell and 
Hutchinson in a paper " On the Effect of Partial Sterilisation of Soil on the 

Production of Plant-Food." t 

In working with these soil-protozoa, so long as one was confined to culture methods 
it was obviously impossible to decide how far any given culture of protozoa gave a 
true picture of the life in a soil, and how far the organisms in the culture were simply 
derived from cysts. There were, of course, certain indications, as one of us has 
pointed out in a previous paper, that certain faunas were connected with certain soils, 

'■ * "Ueber die Verbreitung der Siisswasserprotozoen durch die Luft," 'Archiv fur Protistenkunde/ 
Band 28. 

t ' Journal of Agricultural Science,' vol. 3, Part II (1909). 



80 MESSES. 0. H. MAETIN AND K. E. LEWIN: SOME NOTES ON SOIL PROT020A. 

but even then one was left absolutely in the dark as to what degree of saturation of 
water was necessary in these soils before the animals found in the cultures could lead 
an active life in the soil. 

By a method which one of us has described in a recent letter to c Nature/ No. 2266, 
vol. 91, 1913, and which is given in detail below, it is possible to obtain results which, 
though minimal, are definite as to the state of the fauna in any given soil at any 
moment. Up to the present the only soils we have examined thoroughly by this 
method are (1) a cucumber bed, and (2) a seedling bed obtained from the layer of soil 
underneath the turf in a neighbouring field and free from any admixture of manure. 

In this paper we propose giving some account of the organisms found in an active 
state in these two soils by this method, together with some further details on the 
life-cycles of these forms in cultures. In a future paper we hope to return to this 
question of the existence of active protozoa in the soil in greater detail. 

We should like to take this opportunity of thanking Dr. Russell for the great 
interest which he has taken in this work. We wish also to thank Prof. Minchin 
for his great kindness in allowing us to write up our results in his department at the 
Lister Institute, and Miss Rhodes for the trouble she has taken over the drawings. 

Methods of Preparation and Culture. 

The only method by which, so far as we are aware, the presence of active protozoa 
in the soils can be satisfactorily demonstrated is one which one of us has recently 
described in a letter to ' Nature/ For this purpose a small quantity of the soil to be 
examined is added to about an equal quantity of a saturated aqueous solution of 
picric acid ; the mixture is then stirred very thoroughly, so that the protozoa which 
are situated on the liquid films between the soil particles are freed. The mixture is 
then allowed to stand for some time (12-24 hours), and it will be found that the 
scum which rises to the surface contains a proportion of the bacteria and protozoa 
of the soil. Cover-slip preparations can then be made by floating cover-slips 
upon the surface of the mixture. For the purpose of this work the cover-slips which 
one of us described in a paper on " A Note on the Protozoa from Sick Soils, with 
some Account of the Life-Cycle of a Flagellate Monad," # are very convenient. The 
cover-slip smears thus obtained can be put in corrosivef or simply washed out in 
70-per-cent. alcohol and then stained and mounted in the ordinary way. If the 
organisms in the soil are scarce it will be found an advantage for purposes of 
searching to over-stain in eosin. 

There can be little doubt that this method is not an ideal one, and that it only 
gives minimal results, still in suitable soils quite good results can be obtained, 
for example, in the cucumber soil, the fauna of which is described below, up 
to 150 Amoebae have been obtained on a single film. It is to be hoped, however, 

* <Koy. Soc. Proc.,' B, vol. 85. 

t Vide Mann's * Physiological Histology/ p. 74. 



MESSES. 0. H. MARTIN AND K. R. LEWIN: SOME NOTES ON SOIL PROTOZOA. 81 

that in the near future some better method will be found for dealing with this 
problem, more particularly in poor clay soils, though at present we must confess it is 
very hard to discover one. 

It would we feel be premature at present to attempt a formal list of the culture 
media on which soil protozoa flourish. In all cases of cultures of soil protozoa, so far 
as we are aware, up to the present, as Vahlkampe clearly insisted in his paper on 
the biology, etc., of Amoeba Umax* the protozoa feed upon the bacteria of the 
culture, and hence almost any culture media on which soil bacteria flourish will 
probably support a large number of protozoa. 

Therefore in those cases in which the expression " pure animal culture" is used we 
only wish to indicate that the culture contained only one form of protozoon, though 
of course it contained large numbers of bacteria. It may of course be possible in the 
future to obtain cultures of some saprozoic protozoa free from bacteria, and in certain 
cases we have found indications that certain Amoebae show a distinct preference for 
certain culture media, though here, again, this effect may be a secondary one due to 
the encouragement of a certain type of bacteria. 

Up to the present we have mainly used solid media for our cultures, as we find 
that they are far more convenient for isolating any given form. We used two types 
of culture media, one an ordinary agar made up of 1000 c.c. meat extract and 
15 grm. of agar ; but we have found a culture medium of Friedberger and Keiter 
described in Kolle and Wassermann's ' Handbuch der pathogenen Mikro- 
organismen/ vol. 1, gives very good results for most soil protozoa; it consists of 
a horse-dung agar made up of three lumps of horse dung and 500 c.c. of water, this 
mixture is boiled for one and a half hours, then filtered through cloth, and finally 
about 8 grm. of agar is added. In many cases where it is used to get a very strong 
growth of protozoa it is advisable to add a small amount of water or dilute albumen 
to the culture plates to about a depth of 2 mm. This addition of water seems 
to obviate the vacuolated appearance which some workers have noted as characteristic 
of culture Amoebae on plates. 

The stock cultures are made up by adding a little soil directly to the plates. If 
these stock cultures are examined from time to time it will be found that in any 
given culture there is a more or less definite succession of animal forms. By 
selecting the time and method of culture it will probably be found possible to 
get pure animal cultures of any of these forms. 

In this connection it may be interesting to note the results obtained from two 
cultures which would seem to indicate that the protozoa in the soil are to a great 
extent feeders on bacteria, and that the number of bacteria in the mud from ordinary 
fresh water pools does not seem to offer equal scope for true feeders on bacteria. 
Two cultures were made from the cucumber soil on manure agar to which, in place 
of water, Molisch's solution was added. In these cases the algae got the upper hand 

* i Arch. Protistenk./ vol. 5. 
VOL. CCV. — B. M 



82 MESSRS. 0. H. MARTIN AND K. R. LEWIN: SOME NOTftS ON SOIL PROTOZOA. 

of the bacteria on the culture, and possibly as a result of this the culture from 
a protozoon point of view was a very poor one. The counter-experiment was made 
with some of the cultures of Amoeba proteus, which were placed with some mud on 
manure-agar plates, and here the bacteria did not develop to anything like the 
extent to which they would have done from an ordinary soil culture, and none of the 
ordinary soil protozoa were found. 

Cucumber Bed. 

In July, 1913, through the kindness of a grower in the neighbourhood of 
Eothamsted we had an opportunity of examining a sick cucumber border. In order 
to give a fair picture of the very special conditions prevailing in these cucumber beds 
we have thought it well to give the history of this particular border bed from 
its origin. It was made up in February, 1912, of one part by volume of light 
pasture soil, one part of heavy pasture soil, and two parts by volume of horse manure. 
During the season (April to June 15) the beds were either mulched and given in 
alternate weeks a small dose of chemical food or were given a dressing of soil, cow- 
dung, and soot. After June 15 the same treatment was applied with longer 
intervals. Towards the end of the year two- thirds of the bed was removed and 
replaced by horse manure. On December 27, 1912, the bed was steam' sterilised 
(20 minutes on a steam grid) and then treated as in the previous year. The water 
content on the date on which we first examined the bed (August 1, 1913) was in 
the top layer 62 per cent, by weight, and in the bottom layer 55 per cent. On 
October 7, 1913, the water content was 57 per cent, in a sample from the bottom. 
Probably the water content does not vary much, in these beds. At first sight this 
water content may seem high to those who have had no experience of estimations of 
soil moisture, but the soil at the time of examination was only damp to the touch 
and there was no sign of excessive water. 

Preparations were made of the fresh soil by the picric acid method described above, 
and the following organisms were discovered in the trophic condition on the films so 
prepared : — 

1. Euglypha, ?sp. (Plate 5; fig. 1). 5. Flagellate, ?gen. (Plate 5, fig. 3). 

2. Trinema, ? sp. 6. Chilodon sp. (Plate 5, fig. 2). 

3. Vahlkampfia soli, n. sp. (Plate 5, fig. 10). 7. Ciliate, ?gen. 

4. Amoeba cucumis, n. sp. (Plate 6, fig. 20). 8. Ciliate, ?gen. 

Of these the thecamoebae were probably present in this soil in considerable numbers, 
more than is indicated by the number of forms obtained on the films. 

This is probably due to the fact that under the most favourable circumstances it is 
rather difficult to get good preparations of these thecamoebse from a numerical point 
of view by the cover-slip method. 

The Amoebae were present on the films in large numbers, e.g., up to 150 good 



MESSES. C. H. MARTIN AND K. R. LEWIN: SOME NOTES ON SOIL PROTOZOA. 83 

specimens in an active trophic condition, with large numbers of ingested bacteria, 
were counted on a single film. These were probably the dominant protozoa in this 
soil during the time it was under examination. 

The flagellates were very rare on these films, and this was rather curious, as in 
cultures from this soil they were very numerous (Prowazekia and Copromonas, 
etc.). It would seem that this result can be explained by one of the two following 
hypotheses : — 

(a) The films gave a true picture of the life in this soil, and flagellate forms were 
not present in large numbers in the trophic condition, possibly because the water- 
content of the soil was not large enough to allow them sufficient liberty for long free 
paths ; or 

(b) The flagellates were present in an active condition, but the films did not give a 
true picture, because the long flagella might have become entangled in the soil 
particles, and thus have prevented the flagellates being carried to the surface-film. 

At present w r e are inclined to accept the former of these views, because in 
preparations made in the above manner from other soils we have found fair numbers 
of flagellates in the trophic phase. 

The ciliates in these preparations are also very rare, and here it seems probable 
that their scarcity on the films represents fairly the state of affairs in this soil. It 
is possible that in absolutely saturated soils ciliates may play an active part as a 
bacterial check, but it is difficult to believe that they can exercise an important 
rdle in a sick soil : bed like the one under examination. On three occasions ciliates 
were found in fresh soil-smears : one appeared to be a Chilodon (Plate 5, fig. 2), and 
the two other forms have not been identified with any certainty. 

Of these forms we only propose to deal at length with the two Amoebae, since not 
only were they probably the most important checks on the bacteria in this soil at the 
time of examination, but they also gave the most interesting results on the cultures. 

Vahlkampfia soli (n. sp.). 

To judge from the evidence of the fresh films, this must have been the dominant 
form in the cucumber bed during the month of August. Although this animal agrees 
in many important points with the Amoeba Umax of Vahlkampf's classical paper, yet 
there are certain small points of difference which seem to justify the formation of a 
new species for the reception of these forms. 

In life Vahlkampfia soli is a very active form, showing the characteristic 
movements of an amoeba of the Umax group. In progression a single, large 
pseudopodium, composed almost entirely of ectoplasm, is usually thrown out, and the 
granular entoplasm seems to stream into it. Very often the posterior end shows a 
characteristic tufted appearance, and it is interesting to note that this appearance is 
often found in animals on fresh smears (Plate 5. fig. 10), showing that these forms 
were actively moving through the soil at the time they were fixed by the addition of 

M 2 



84 MESSRS. C. H. MARTIN AND K. R. LEWIN: SOME NOTES ON SOIL PROTOZOA. 

picric acid. A contractile vacuole is present, and is usually discharged at the 
posterior end. The period of active life on these culture-plates at 18° C. seems to be 
about 3-4 days. In life certain highly refractile granules are fairly frequent in the 
endoplasm, especially of young forms. In fresh smears prepared from this cucumber 
soil by the picric acid method, the trophic stages of this animal were very 
abundant during the month of August. The forms thus found were usually very 
active, and contained large numbers of ingested bacteria (Plate 5, fig. 10). On some 
smears indications of division were found (Plate 5, fig. 4), thus clearly showing that 
Vahlkampjia soli flourished in this soil at this date. Vahlhampfia soli was found to 
do fairly well on manure-agar cultures with water, and we also tried plates made up 
of a straw decoction, which had been recommended by Vaolkampf. In stained 
forms from young cultures the nucleus is a spherical body, with a large, darkly- 
staining karyosome of rather elliptical form. The membrane is a fairly definite 
structure, and ranged upon it there appear to be a number of masses staining with 
eosin. These forms from young cultures corresponded in every way with the forms 
found in the fresh films, and it would seem that this appearance of the nucleus is 
correlated with the intense reproductive activity characteristic of this stage. In 
older cultures the karyosome is rounder, smaller, and denser ; some of the stages of 
division appear to resemble very closely stages figured by Glaser in the division of 
Amoeba tachy podia, and pfobably some of the stages figured by Aragao for Amoeba 
diplomitotica. 

It is highly probable, however, that there are a very large number of amoebae for 
which this statement will be found true. 

As regards the behaviour of the cytoplasm during division, it is perhaps worth 
noting that the rounding up which Glaser has stated to be always characteristic of 
dividing amoebae is not seen in this form, and that the stage of nuclear division does 
not seem to bear an absolutely fixed relation to the progress of the cytoplasmic 
division. 

The nuclear division of this amoeba belongs to that very difficult type, for which 
Nagler has invented the term of promitosis. In the early stages of division 
the karyosome swells and becomes very elliptical, and the mass of the chromatin 
then gets concentrated at either pole of the elongated karyosome. There can 
be no doubt that the division of the great mass of chromatin contained in the 
resting nucleus is effected by this simple means. The question of chromosomes in 
this division is an extraordinarily difficult one, and one cannot help feeling that in 
divisions of this kind the result arrived at depends much upon the technique adopted, 
and in many cases it would appear that too much reliance is placed on iron 
hematoxylin preparations alone. In this case we have tried the combined results 
obtained from iron hsematoxylin and hsemalum preparations, though we decided, as a 
matter of caution only, to figure the hsemalum preparations. At a slightly later 
stage it would appear that the median portion of the karyosome becomes broken on 



MESSRS. G. H. MARTIN AND K. R. LEWIN: SOME NOTES ON SOIL PROTOZOA. 85 

one side into a mass of irregular granules (Plate 5, fig. 11). This stage seems to 
correspond in some respects with that figured by Glaser for Amoeba tachypodia, but 
in this case he is inclined to refer the origin of these granules to extra- karyosomic 
chromatin. The dumb-bell-shaped karyosome is now broken through (Plate 5, 
figs. 12 and 13), and the two polar masses gradually pass to the opposite extremities of 
the dividing animal (Plate 5, fig. 14). The connecting bar, which seems to consist of 
a mixture of the chromatin granules mentioned above and an achromatic mass, 
becomes gradually thinner at its middle, and, as by this process chromatin granules 
are brought much closer together, it becomes at the same time much more con- 
spicuous (Plate 5, fig. 15). During this stage, for some unknown reason, the 
chromatin becomes massed in three volumes, viz. the two polar masses, and the 
median portion of the connecting bar, so that the characteristic appearance shown in 
Plate 5, fig. 15, is originated by two polar masses, an intervening clear space, and a 
darkly-stained central bar. The daughter nuclei now become separated through the 
snapping of this connecting bar, and in the reconstruction of the nuclei it would 
appear that the wedge-shaped mass derived from the connecting bar loses its 
chromatin in the form of irregular granules, which fuse with the karyosome (Plate 5, 
fig. 16). It will be obvious from this account that, though we differ with Glaser 
over some slight points of detail, yet in this form also we can find no trace of the 
existence of a centriole. 

In life the cysts of these amoebae are very characteristic objects on the culture- 
plates. They have an outer gelatinous coat, to which foreign objects become 
attached, and an inner resistant wall of a yellowish colour. The cysts are 
frequently grouped together in a culture, and this feature appears to be somewhat 
important, as upon it some of the earlier workers, e.g. , Zopf, place similar forms in 
the group of Mycetozoa. In his account of Copromyxa protea in 'Die Pilzthiere 
oder Schleimpilze/ Zopf states on p. 133 : — 

" Da auf trocknen Mistculturen die Amoeben gewohnlich nicht alle neben, sondern 
zum Theil liber einander kriechen, so entstehen die eingangs erwahnten Sporen- 
haufchen (Sort) (fig. 31,1, II)." 

The mature cysts are extraordinarily difficult to stain. It seems, however, from 
early stages of encystation, such as is shown in Plate 6, fig. 19, that there is a 
single central nucleus, and we have no reason to suppose that the cyst is anything 
but a protective cyst. Under ordinary cultural conditions this form emerges from 
the cysts as a typical ^'max-amoeba ; in the process of excystation the cyst swells 
considerably and the amoeba can be seen moving round actively in the cyst for some 
time before it emerges through a small opening in the cyst wall. 

The Flagellate Form.— In 1910, Wasielewski and Hirschfeld pointed out the 
appearance of certain conditions of a flagellate stage of fo'max-amoebse, and more 
recently Whitmore and Wherry have described such stages. In the present case 



86 MESSRS. C. H. MARTIN AND K. R. LEWIN: SOME NOTES ON SOIL PROTOZOA. 

it was found that, if a plate of manure- agar were heavily infected with the cysts and 
covered to a depth of about 2 mm. with tap- water containing 0*25 per cent. NaCl 
and 0*05 per cent. MgS0 4 , after about 16-20 hours' incubation at 23° C, flagellate 
forms of this amoeba appear sporadically. On three occasions an example of the 
flagellate was continuously watched under a 2*5 mm. water-immersion objective, and 
was seen to become amoeboid, lose its flagella by absorption, and turn into a typical 
specimen of the amoeba. Permanent preparations of this transition have also been 
obtained (Plate 6, fig. 18). The flagellate stage (Plate 5, fig. 17) is generally ovoid, 
with an oblique anterior end, but some specimens are markedly piriform, the pointed 
end being anterior. The two flagella arise anteriorly and are each about equal in 
length to the body. The nucleus is situated near the insertion of the flagella, and 
in stained preparations a fibril connecting the nuclear membrane with the common 
root of the flagella can be detected. 

A contractile vacuole is present, occurring laterally in the posterior third of the 
body. It arises as a group of small vacuoles, which coalesce to one large one that 
bulges out the body-wall and eventually bursts like a bubble. Its period is 
two to three minutes. 

The cytoplasm is fairly homogeneous, and at the oblique anterior end there seems 
to be a slight differentiation recalling ectoplasm. The flagellate is flexible, but shows 
no tendency to put out pseudopodia. 

Movements. — These are quite rapid, translocation being effected by a steady 
shouldering movement, during which the animal rotates on its long axis. The 
flagella are hardly to be made out unless the flagellate be momentarily at rest. They 
appear to whip right back to the sides of the body. 

Feeding. — Bacteria are ingested at the oblique anterior surface, where a sorb of 
ectoplasmic differentiation is seen. They pass by the nucleus and are finally digested 
in the median and posterior parts of the body. 

Transition to Amoeba. — The flagellate remains in one place, and the outlines of its 
body appear slightly wavy. Then it becomes roughly spherical and protrudes 
pseudopodia in all directions. Almost at once the spherical form is lost and a 
typical amoeboid appearance is attained. The flagella lose their anterior position 
and become lateral, or often terminal, but for about seven minutes emerge always in 
the neighbourhood of the nucleus, to which they are attached. After this tinje the 
connection presumably breaks, and the position of the nucleus is no index to that of 
the flagella, which continually tend towards the hind end at any moment. Shortly 
after attaining independence, the flagella are active only in a minor degree, and soon 
become motionless. About 15 minutes (hanging drop preparation ; less under 
cover- slip) after the first signs of change, the flagella are absorbed by a pseudopodium 
which pushes out close to their origin, thrusts them aside, and flows along them and 
engulfs them. 

This process may go some way and then suffer reversal. On one occasion a 



MESSES. 0. H. MARTIN ANb K. fe. LEWIN: SOMlS NOTES ON SOIL PROTOZOA. 8? 

flagellate was seen to round up, protrude pseudopodia, and take on typical amoeboid 
form, and after 2^ minutes gradually to recover the definite flagellate shape. After 
this it was watched continuously for 25 minutes, and then showed no signs of 
transition. In this instance of the flagellates attaining the amoeboid form, the 
flagella did not lose their connection with the nucleus. 




Text-fig. 1. — -Transformation of Flagellated into Amoeboid Stage of V. soli. 

1, Flagellate stage; 2, start of transformation; 3, 1 minute after start; 4, 2 minutes; 5, 3 minutes; 

6, 7 minutes; 7, 9 minutes; 8, 12 minutes; 9, 15 minutes; 10, 17 minutes. 

The significance of these flagellate stages is at present unknown, but whether their 

appearance forms grounds for removing the limax- ( a,modbdd from the group of true 

Amoebae and placing them amongst the Proteomyxa is a question that future work 

must decide. 

Amoeba cucumis (n. sp.). 

Amoeba cucumis was also met with in the cucumber soil, and it appeared in the 
cultures of manure-agar plates associated with Vahlkampjia, soli. It could be readily 
distinguished from the Vahlkampfia form in life since it is a very sluggish form with 
very characteristic pseudopodia. Dividing forms were most frequently found on 



88 MESSES. C. H. MARTIN AND K. H. LEWIN: SOME NOTES ON SOIL PKOTOZOA. 

cultures three to four days old, and cysts were not frequent until the eighth or 
ninth day. Active forms associated with cysts have, in fact, been met with in 
cultures over a month old, though these forms were then of rather small size. In 
young cultures the typical appearance seems to be an almost spherical form with 
rather fine radiating pseudopodia composed almost entirely of ectoplasm. Frequently, 
however, forms are met with in which a kind of wave of ectoplasm is thrown out. 
As far as can be seen there is no definite contractile vacuole. This form is not nearly 
such a typical devourer of bacteria as Vahlkampjia soli, since it not infrequently 
ingests small flagellates, cysts, etc. 

The cytoplasm has a tendency to take up stains rather deeply. The nucleus is 
almost central, it consists in young cultures of a large, darkly staining karyosome 
separated by a considerable space from a distinct membrane, and in this space small 
particles, composed apparently of chromatin, lie, their presence being more noticeable 
in forms from older cultures. The division of this form seems to show many points 
of similarity to that described by Glaser for Amoeba lamellipodia. 

In the early stages of division the karyosome becomes much swollen and the 
cytoplasm of the body becomes very definitely rounded up (Plate 6, fig. 22). The 
karyosome then becomes broken up into a number of small chromatin masses, and 
these become arranged in a plate which may extend over half of the diameter of the 
animal (Plate 6, fig. 23). The chromatin-granules are at first present in a number 
of rows, but later they become arranged in a double row, thus giving rise to two 
distinct equatorial plates (Plate 6, fig. 24). These two plates, which lie in a 
differentiated tract in which we can find no trace of a definite fibril structure, 
become gradually shifted apart and at the same time more concentrated (Plate 6, 
fig. 26). At about this stage the first signs of division are seen in the cytoplasmic 
body. The chromatin-granules now become lumped into masses which coalesce to 
form a new karyosome. An odd feature of this division is the prominence of a thin 
thread connecting the daughter-individuals (Plate G, fig. 28), which seems absolutely 
identical with that figured by Glaser for Amoeba lamellipodia. In many cases 
encystation seemed to be preceded by an association of two individuals correlated 
with some rather complicated nuclear changes ; to these, however, we hope to return 
on a future occasion. The ripe cyst in life is a thin-walled, colourless structure 
(Plate 6, fig. 29). The nucleus lies rather eccentrically, and the cytoplasm is at 
certain stages crowded with darkly staining masses which appear to be extruded by 
chromatin granules. 

In the process of excy station it appears that the cyst- wall is dissolved. For 
example, an excysting form was picked up at 3.30; at this time the cyst-wall was 
quite definite and the nucleus rather elongate. Vacuoles now made their appearance 
at the periphery of the cytoplasm and under the cyst- wall, and by 3.58 the wall 
was completely dissolved and the typical membranous pseudopodia had made their 
appearance. 



MESSRS. C. H. MAETIN AND K. E. LEWIN: SOME NOTES ON SOIL PEOTOZOA. 89 



Seedling Bed. 

In March of this year one of us had an opportunity of examining, through the 
kindness of Mr. Pitt of Abergavenny, the soil of a frame bed in which seedling 
cauliflowers were being grown. The bed was made this year by taking the first spit 
of soil after the turf had been removed from a neighbouring field and mixing it 
with sand and leaf mould ; no manure was added, and the frame was not heated, so 
that the conditions were as different from those in the preceding soil as could be 
imagined. 

The active fauna found by means of the picric acid method was not as rich in the 
number of individuals but far richer in the number of species than the cucumber soil. 
This case seems to present an interesting analogy from a faunistic point of view to 
results obtained on the grass plots at Rothamsted, where, as is well known, the 
untreated plot still gives a large number of species, whereas in some of the plots 
which have received heavy manure of one kind for many years the number of species 
has been cut down to four or five. 

The same phenomenon seems to be shown in rich infusions, in which as a rule one 
or other protozoon gets an upper hand, whereas in ordinary fresh-water pools the 
fauna, though far richer from the point of view of the number of species to be met 
with, is far poorer in the actual numbers of individuals. In fresh preparations of this 
soil, of which only a few were carefully examined, the following were some of the 
protozoa met with in a trophic condition : — 

1. Euglypha, ? sp. (Plate 5, fig. 5). 5. Amoeba, ? sp. (Plate 5, fig. 9). 

2. Chlamydophrys, ?sp. (Plate 5, fig. 6). 6. Amoeba, ? sp. 

3. Amoeba gobanniensis, n. sp. (Plate 5, 7. Flagellate amoeba. 

fig. 7). 8. Bodo caudatus (Plate G, figs. 35, 36, 

4. Amoeba, ? sp. (Plate 5, fig. 8). and 37). 

On cultures from this soil Amoeba diploidea, a monad and Prowazekia sp. ? and 
a small ciliate were also met with. It is probable that a more careful search would 
have revealed Amoeba diploidea in a trophic condition on fresh films of this soil. 

In this paper we only propose to note some of the forms met with in this soil 
with the exception of an amoeba, Amoeba gobanniensis n. sp., which seems of 
especial interest since it is evidently so closely allied to the Amoeba cucumis found in 
the cucumber soil. 

The Chlamydophrys found in this soil seems to be rather an interesting form as it 
differs in respect of its size and behaviour of the pseudopodia from the Chlamydophrys 
which one of us cultured out of a soil two years ago and has kept under culture 
ever since in the hope of being able to confirm Schaudinn's account ot the life-cycle 
of this form. Further details of this form will probably be published in a future 
paper. 

VOL. CCV. — B. N 



90 MESS&S. C. It. MABTW AND K. K. LEWlN : SOME NOTES ON SOIL PftOTO^OA. 

An amoeboid form which was rather interesting from the readiness with which it 
assumed flagellate condition was met with in this soil. It seemed to divide in the 
amoeboid condition. 

A relatively large amoeba was also met with in fresh films and in cultures from 
this soil. 

A Bodo, which we are inclined to identify with the Bodo caudatus of Dujardin, 
was met with both on the fresh smears and in the cultures. As will be remembered, 
this species is characterised by a peculiar method of multiple division, and it is, as far 
as we are aware, the only flagellate of a Bodo type for which this method of division 
has been described (Plate 6, figs. 35, 36, and 37). 

From the point of view of nomenclature it is interesting to observe that this 
Bodo has a typical kinetonucleus, and this would seem to emphasise a point already 
brought out by Alexeieff as to the very unsatisfactory state of the classification of 
the genera Bodo and Prowazekia. Some stages of the division of this form are 
shown. 

The behaviour of the nucleus seems rather complicated and will be dealt with more 
fully by one of us in a future paper in the ' Zoologischer Anzeiger.' 

Amoeba gobanniensis, n. sp. 

This amoeba was met with on the fresh films of the seedling soil and on cultures 
made from it on manure-agar and water. 

Amoeba gobanniensis is again extraordinarily like the Amoeba lamellipodia 
described by Glaser (16) and the previously described Amoeba cucumis, but here 
again there are certain distinctive features which seem to show the necessity for the 
formation of a new species, in order to avoid the danger of including two different 
forms under the same specific name. 

In life this amoeba is a very sluggish form, and is readily recognised by the 
extraordinary development of its ectoplasm. The characteristic prickle pseudopodia 
of Amoeba cucumis (cf. Plate 6, fig. 21) are absent in this form, in which the resting 
form is characterised by a large plate-like ectoplasmic pseudopodium surrounding the 
animal. In movement a long axial pseudopodium from the entoplasmic core seems to 
be thrown out by the animal. 

The nucleus in the stained form consists of rather a small karyosome, between 
which and the membrane lie a number of irregular granules. 

The behaviour of this animal in division recalls in many points the division of 
Amoeba cucumis, particularly in the last stage of division, in which the dividing 
products remain for some time connected by a narrow thread of protoplasm (Plate 6, 
fig. 34). In examining a large number of division stages of both these forms, there 
seem to be certain constant points of difference. Firstly, the dividing Amoeba 
gobanniensis never becomes so spherical as the Amoeba cucumis, and it is always 



MESSRS. C. H. MARTIN AND K. R. LEWIN: SOME NOTES ON SOIL PROTOZOA. 91 

characterised by the behaviour of the ectoplasm. Secondly, the band of chromatin 
granules in the early stage of division is not nearly so broad as in the corresponding 
stage of Amoeba cucumis, and in the later stages the spindle seems always slightly 
smaller. Thirdly, there is a more definite indication of fibrous structure in the 
spindle of Amoeba gobanniensis (Plate 6, fig. 32). 

The stages leading up to encystation seem to show similar nuclear complications to 
those that have been noticed in Amoeba cucumis, and the cyst seems to be very 
similar. 

Conclusions. 

It seems generally agreed that further examination of the Amoebae will necessitate 
the splitting up of the genus Amoeba into a number of genera. The first and 
much needed step in this reform has already been taken by Chatton (13), by the 
formation of the genus Vahlhampfia for the group of Umax amoebae. Whether 
it would not be better to put this genus and all the other amoebae which show a 
flagellate stage in their life-cycle apart from the gamete stage into the group 
Proteomyxa seems to us an open question. There can be no doubt that in this genus 
Vahlkampjia a number of quite definite species are included which can probably be 
best separated by minute differences in the behaviour of the nucleus during division. 
It will probably be found necessary in the same way to form another genus for the 
lamellipodia group of amoebae, which would again have to be broken up into a 
number of species in a similar manner. 

The main purpose of this introductory paper has not, however, been the study of 
these amoebae from a specific point of view, so much as the proof which we hope to 
have brought of the existence of a relatively frequent trophic Protozoan fauna in 
certain soils and the rough indication of some possible methods of dealing with this 
fauna. How far this fauna under certain conditions exercises a deleterious influence 
on plant growth is rather a question for the agriculturist than the zoologist. 

The startling success in the Lee Valley of the treatment of sick soils by partial 
sterilisation, introduced by Russell, would seem to present a very strong argument 
in favour of the view that these protozoa do exercise an important influence on plant 
growth in these soils. We have, by means of the method described above, been able 
to establish the occurrence of a trophic Protozoan fauna in certain field soils that we 
have examined, and to this question we hope to return in a future paper. 



N 2 



92 MESSRS. C. H. MARTIN AND K. R. LEWIN : SOME NOTES ON SOIL PROTOZOA. 



LITERATURE. 

1. Alexeieff, "La Division nucleaire et 1'Enkystement chez quelques Amibes, 

I-III," ' Compt. Rend. Soc. Biol./ vol. 70. 

2. Idem, " Sur le Stade Flagelle dans Involution des Amibes limax," ' Compt. Rend. 

Soc. Biol./ vol. 72 (1912). 

3. Idem, " Sur les Caracteres Cytologiques et la Systematique des Amibes du 

groupe Limax (Ncegleria nov. gen. et Hartmannia nov. gen.) et des 
Amibes Parasites des Vertebres (Proetamoeba nov. gen.)," ' Bull. Soc. 
Zool. France/ vol. 37 (1912). 

4. Idem, " Quelques Remarques Complementaires sur la Systematique des Amibes 

du groupe Limax," ' Bull. Soc. Zool. France/ vol. 37 (1912). 

5. Idem, " Systematisation de la Mitose dite 'primitive/" 'Arch. Protistenk./ 

vol 29 (1913). 

6. Aragao, " Amoeba diplomitotiea," ' Mem. do Inst. Oswaldo Cruz/ vol. 1 (1909)- 

7. Behla, ' Die Amoben. insbesondere vom parasitaren und kulturellen Stand- 

punkte.' Berlin, 1898. 

8. Beyerinok, M. W., " Kulturversuche mit Amoeben auf festen Substraten," 

'Centralb. f. Bakt. u. Parasit., Jena/ vol. 19 (1896), and vol. 21 (1898). 

9. Butschli, " Protozoa," ' Bronns Thierreich.' 

10. Brodsky, " Division and Encystment of Amoeba hyalina (Davy)," 'Biol. Zeitsch./ 

vol. L (1910). 

11. Chatton, "Sur quelques genres d' Amibes libres et parasites," ' Bull. Soc. Zool. 

France, vol. 37 (1912). 

12. Idem, " La Structure du Noyau et la Mitose chez les Amcebiens," 'Archives de 

Zool. Exper./ vol. 5 (1910) 

13. Chatton and Lalung-Bonnaire, "Une Amibe Limax (Vahlkampfia, N . G.) 

dans Tintestin humain," ' Bull. Soc. Path. Exot./ vol. 5. 

14. Dangeard, " fitudes sur le ddveloppement des Organismes inferieurs," ' Le 

Botaniste/ vol. 11 (1910). 

15. Frosch, P., " Zur Frage der Reinzuchtung der Amoeben," ' Centralb. f. Bakt. 

u. Parasit./ vol. 21. 

16. Glaser, " Uber die Teilung einiger Amoben," ' Arch. Protistenk.,' vol. 25 

(1912). 
'17. Idem, " Kernteilung Encystierung und Reifung von Amoeba mira," 'Arch. 

Protistenk.,' vol. 27 (1912). 

18. Hautmanx and Chagas, " Uber die Kernteilung von Amoeba hyalina (Davy)," 

' Mem. Inst. Oswaldo Cruz./ vol. 2 (1910). 

19. Hartmann, M., and Nagler, K., " Copulation bei Amoeba diploidea, etc.," 

* Sitzb. d. Ges. d. Naturf. Freunde.' Berlin, 1908. 



MESSES. 0. H. MARTIN AND K. R. LEWIN : SOME NOTES ON SOIL PROTOZOA. 93 

20. Nagler, " Entwieklungsgeschichtliche Studien liber Amoeben," ' Arch. 

Protistenk./ vol. 15 (1909). 

21. Idem, " Studien liber Protozoen aus einem Almtumpel— Amoeba Hartmanni" 

6 Arch. Protistenk./ vol. 22(1911). 

22. Idem, "Die Kern- und Centriolteilung bei Amoben," 'Arch. Protistenk./ 

vol. 26 (1912). 

23. Puschkarew, " Uber die Verbreitung der Slisswasser- protozoen durch die Luft," 

' Arch. Protistenk./ vol. 28 (1913). 

24. Vahlkampf, E., " Biologie und Entwicklungsgechichte von Amoeba Umax" 

6 Arch. Protistenk./ vol. 5, p. 167 (1905). 

25. Walker, " Die Technik der Amoebenzuchtung," ' Centralb. flir Bakter./ vol. 50 

(1911). 

26. Wasielewski and Hirschfeld, " Untersuchungen li. Kulturamoben," 'Abh. 

Heidelberg. Akad. Wiss. Math.-naturw. Kl/ (1910). 

27. Wherry, " Studies on the Biology of an Amoeba of the Limax group," ' Arch. 

Protistenk./ vol. 31 (1913). 

28. Whitmore, " Studien liber Kulturamoben aus Manila," 'Arch. Protistenk./ 

vol. 23 (1911). 

29. Zopf, "Die Pilztiere oder Schleimpilze," ' Encyclopadie der Natur wiss./ 

Breslau, 1885. 



DESCRIPTION OF PLATES. 

Plate 5. 

Fig. 1. — Euglypha sp. from fresh preparation of cucumber bed. 
2. — Chilodon sp. from fresh preparation of cucumber bed. 
3, — Flagellate from fresh preparation of cucumber bed. 
4. — Dividing Vahlkampfia soli from fresh preparation of cucumber bed. 
5. — Euglypha sp. from fresh preparation of seedling bed. 
6. — Chlamydophrys sp. from fresh preparation of seedling bed. 
7. — Amoeba gobanniensis from fresh preparation of seedling bed. 
8. — Amoeba sp. from fresh preparation of seedling bed. 
,, 9. — Amoeba sp. from fresh preparation of seedling bed. 

Vahlkampfia soli (n. sp.). 

(All the drawings of this form were made under Zeiss 2 mm. apoch. + 18 comp. oc.) 

Fig. 10. — Vahlkampfia soli from fresh preparation of cucumber bed. 
Figs. 11-16. — Stages in division of Vahlkampfia soli. 
Fig. 17. — Flagellated stage of Vahlkampfia soli. 



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94 MESSRS. C. H. MARTIN AND K. R. LEWIN: SOME NOTES ON SOIL PROTOZOA 

Plate 6. 

Vahlkampfia soli (n. sp.). 

Fig. 18. — Transition to amoeboid stage (whole length of flagella not shown). 
„ 19. — Cyst of Vahlkampfia soli. 

Amoeba cucumis (n. sp.), 

(Zeiss 2 mm. apoch. + 12 comp. oc.) 

Fig. 20. — Amoeba cucumis from fresh preparation of cucumber bed. 

„ 21. — Amoeba cucumis from young culture. 
Figs. 22-28. — Stages in division of Amoeba cucumis. 
Fig. 29. — Cyst of Amoeba cucumis. 

Amoeba gobanniensis (n. sp.). 

(Zeiss 2 mm. apoch. + 12 comp. oc.) 

Fig. 30. — Amoeba gobanniensis from culture. 

Figs. 31-34.- — Stages in division of Amoeba gobanniensis. 

Bodo caudatus. 

(Zeiss 1*5 mm. apoch. + 18 comp. oc.) 

Fig. 35. — Bodo caudatus. 
„ 36. — Multiple division of Bodo caudatus. 
„ 37. — Multiple division of Bodo caudatus, later stage. 



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Plate 5. 

Fig. 1. — Euglypha sp. from fresh preparation of cucumber bed. 
2. — Chilodon sp. from fresh preparation of cucumber bed. 
3. — Flagellate from fresh preparation of cucumber bed. 
4. — Dividing Vahlkampfia soli from fresh preparation of cucumber bed. 
5. — Euglyp)ia sp. from fresh preparation of seedling bed. 
6. — Chlamydophrys sp. from fresh preparation of seedling bed. 
7. — Amceba gobanniensis from fresh preparation of seedling bed. 
8. — Amoeba sp. from fresh preparation of seedling bed. 
9. — Atnceba sp. from fresh preparation of seedling bed. 



Vahlkampfia soli (n. sp.). 

(All the drawings of this form were made under Zeiss 2 mm. apoch. + 18 comp. oe.) 

Fig. 10. — Vahlkampfia soli from fresh preparation of cucumber bed. 
Figs. 11-16. — Stages in division of Vahlkampfia soli. 
Fig. 17. — Flagellated stage of Vahlkampfia soli. 






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Plate 6. 
Vahlkampfia soli (n. sp.). 
Fig. 18. — Transition to amoeboid stage (whole length of flagella not shown). 
,, 19. — Cyst of Vahlkampfia soli. 

Amceha cucumis (n. sp.), 
(Zeiss 2 mm. apoch. + 12 comp. oc.) 
Fig. 20.— Amceba citcumis from fresh preparation of cucumber bed. 

,, 21. — Amceba cucumis from young culture. 
Figs. 22-28.— Stages in division of Amceba cucumis. 
Fig. 29. — Cyst of Amceba cucumis. 

Amceha gohanniensis (n. sp.). 
(Zeiss 2 mm. apoch. + 12 comp. oc.) 
Fig. 30. — Amceba gohanniensis from culture. 
Figs. 31-34. — Stages in division of Amoeba gohanniensis. 

Boclo caudatus. 
(Zeiss 1*5 mm. apoch. + 18 comp. oc.) 
Fig. 35. — Bodo caudatus. 
„ 36. — Multiple division of Bodo caudatus. 
,, 37. — Multiple division of Bodo caudatus, later stage.