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PROTOZOA 

IN BIOLOGICAL 

RESEARCH 



PROTOZOA 

IN BIOLOGICAL 

RESEARCH 



Edited by 

GARY N. CALKINS 

and 

FRANCIS M. SUMMERS 




New York: Morningside Heights 

COLUMBIA UNIVERSITY PRESS 

19 4 1 



Copyright 1941 

COLUMBIA UNIVERSITY PRESS, NEW YORK 

Foreign agents; Oxford University Press, Humphrey Milford, Amen 

House, London, E.C. 4, England, and B. I. Building, Nicol Road, 

Bombay, India; Maruzen Company, Ltd., 6 Nihonbashi, 

Tori-Nichome, Tokyo, Japan 

Manufactured in the United States of America 




PREFACE 

Numerous textbooks of varying degrees of excellence for the study 
of Protozoa are now on the market and should be consulted for a 
general treatment of these unicellular forms. This volume will not fill 
such a need, but has been prepared as a result of a discussion by a 
group of specialists assembled during the summer of 1937 at the 
Marine Biological Laboratory at Woods Hole, Massachusetts, for the 
purpose of ascertaining the best means to stimulate further research 
on these unicellular animals. 

As a result of these discussions it was decided that one good way to 
attain our end would be to have a group of specialists in the field of 
proto2o5logy prepare a work on research in this field, each specialist 
to provide a chapter on the subject in which he is best known, and 
about which he can speak with authority. 

Our first real difficulty was to select a limited number of topics from 
a vast number of possibilities, and then to choose the biologists who, in 
our opinion, were the best men to write these chapters. As can be 
readily imagined, this opened up a long list of difficulties and led to 
many vexatious troubles, but the present work, finally, is the outcome. 

To our very great regret, one of the men chosen — Professor Noland, 
of the University of Wisconsin — who was most enthusiastic over the 
project, has been forced by reason of continued illness, to drop out. 
The loss of others, on the plea of pressure of work, and so forth, has 
also depleted our ranks. But the remainder have completed their labors, 
have come through, and the results here speak for their continued in- 
terest and assiduity. 

While the arrangement of our material does not make a great 
deal of difference, some order nevertheless is advantageous, and we 
have therefore arranged the chapters, according to the general character 
of their materials, into groups having a more or less common subject 
matter. 

Gary N. Calkins 
Francis M. Summers 
New York City 
January 2, 1941 



ACKNOWLEDGMENTS 

We are indebted to the American publishers of the following 
books or periodicals for permission to use figures or other illustrations 
which are reproduced in this book. In a few cases these have been 
slightly modified by the contributors. Also we are under obligation to 
numerous foreign publishers who, for obvious reasons, we have been 
unable to ask for permission. 

American Journal of Hygiene 

Figures 188, 191, 192 
Biological Bulletin 

Figures 5, 10, 13, 74, 81, 82, 84, 96, 104, 181, 183, 185, 186, 187, 
199A, 199B, 223 
Biology of the Protozoa by G. N. Calkins, 1933. Lea and Febiger Co. 

Figures 1, 2, 3 A, 3B, 9 
Genetics 

Figures 166, 168 
Journal of Animal Behavior, Henry Holt and Co. 

Figure 103 
Journal of Cellular and Comparative Physiology, Wistar Institute 

of Anatomy and Biology 

Figures 119, 120, 121, 122, and Table 2 
Journal of the Elisha Mitchell Scientific Society 

Figures 202A, 202B 

Journal of Experimental Medicine, Rockefeller Institute for Medical 

Research 

Figures 189, 190 
Journal of Experimental Zoology, Wistar Institute of Anatomy and 

Biology 

Figures 14, 94, 97, 98, 102, 105, 106, 149, 156, 162, 164, 179, 182 
Journal of Morphology, Wistar Institute of Anatomy and Biology 

Figures 83, 91, 93, 154, 157, 160, l6l, 163 



viii ACKNOWLEDGMENTS 

Journal of Preventative Medicine, John McCormick Institute for In- 
fectious Diseases 
Figure 193 

Light and Behavior of Organisms by S. O. Mast, 1911. John Wiley 
and Sons, Inc. 
Figures 99, 107 

Physiological Zoology, University of Chicago Press 
Figures 95, 113, 126, 180 

Publications of the Carnegie Institute of Washington 
Figure 214 

Science 

Figure 123 

Transactions of the American Microscopical Society 
Figures 36, 37 

University of California Publications in Zoology, University of Cali- 
fornia Press 

Figures 15, 16, 39, 49, 50, 53, 54, 55, 70, 85, 86, 87, 88, 89, 
146, 148, 150, 151, 196, 219, 225 

United States Geological Survey of the Territories 
Figure 221 

Finally, it is a pleasure to recognize the care and patience on the part 
of the staff of the Columbia University Press in the preparation and 
distribution of this v^ork. While all members have worked diligently 
and harmoniously with us, we are particularly indebted to^ Miss Georgia 
W. Read, Miss Ida M. Lynn, and Miss Eugenia Porter for the meticu- 
lous care with which they have sought to unify and, in some cases, to 
clarify the manuscripts on subjects unfamiliar to them. 

The Editors 
New York City 
January 2, 1941 



CONTENTS 

List of Abbreviations xxvii 

I. General Considerations By Gary N. Calkins, Pro- 
fessor Emeritus of Protozoology in Residence, Co- 
lumbia University .... 3 

Life and vitality — Fundamental and derived organiza- 
tion — Some ecological considerations — Some historical 
facts — The use of cultures — Factors influencing longev- 
ity — Changes with metabolism — Reorganization of the 
macronucleus and other derived structures in Ciliata — 
Reorganization by endomixis and by conjugation — Lit- 
erature cited 

IL Some Physical Properties of the Protoplasm of the 
Protozoa By H. W. Beams and R. L. King, State 
University of loiva 43 

Introduction — Properties of protoplasm as exhibited in 
Amoeba— Colloidsil nature of protoplasm — Consist- 
ency — Surface properties — Specific gravity or density — 
Optical properties — Structural properties — Literature 
cited 

in. Cytoplasmic Inclusions By Ronald F. MacLennan, 

Oberlin College Ill 

Mitochondria — The vacuome hypothesis — Digestive 
granules — Segregation granules — Golgi bodies — Excre- 
tory granules — Lipoid reserves — Carbohydrate reserves — 
Protein reserves — External secretion — The granular com- 
plex — The continuity of cytoplasmic granules — The 
classification of cytoplasmic granules — Comparison with 
cells of the Metazoa — Literature cited 

IV. Fibrillar Systems in Ciliates By C. V. Taylor, Stan- 
ford University 191 

Introduction — Examples of fibrillar systems — Structural 



53683 



CONTENTS 

analysis — Interpretation — Fibrillar systems of other 
ciliates — Holotricha — Heterotricha - — Oligotricha — 
Hypotricha^Conclusions — Literature cited 

V. Motor Response in Unicellular Animals By S. O. 
Mast, Director, Zoological Laboratory, Johns Hop- 
kins University 271 

Introduction — Responses to light — Rhizopods — Flagel- 
lates — Ciliates — Colonial organisms — Responses to elec- 
tricity- — Rhizopods — Flagellates — Ciliates — Colonial or- 
ganisms — Responses to Chemicals — Rhizopods— Ciliates 
— Literature cited 

VI. Respiratory Metabolism By Theodore Louis Jabn, 

State University of lotva 352 

Purposes of studying respiration — Methods of measuring 
aerobic respiration — Aerobic respiration — Investigations 
which concern the source of energy — Investigations 
which concern the mechanism of respiration — The 
measurement of anaerobic metabolism and glycolysis — 
Occurrence of anaerobiosis and glycolysis — Why are 
anaerobes anaerobes, and aerobes aerobes? — Oxida- 
tion-Reduction potential versus respiration and growth — 
Literature cited 

Vn. The Contractile Vacuole By J. H. Weatherby, Medi- 
cal College of Virginia 404 

Introduction — The origin of contractile vacuoles — The 
structure of contractile vacuoles — The function of con- 
tractile vacuoles — Contractile vacuoles and the Golgi 
apparatus — Conclusion — Literature cited 

VIII. The Technique and Significance of Control in Proto- 
zoan Culture By George W. Kidder, Brotvn University 448 

Introduction — The problem of protozoan sterilization — 
General material — General methods of sterilization — ■ 
Special methods and manipulations — The importance of 
adequate sterility tests — Establishment of sterilized Pro- 
tozoa in culture — Literature cited 



CONTENTS xi 

IX. Food Requirements and Other Factors Influencing 
Growth of Protozoa in Pure Cultures By R. P. Hall, 

University College, New York University ... 475 

Food requirements of Protozoa — Photoautotrophic nu- 
trition— Photomesotrophic nutrition — Photometatrophic 
nutrition — Heteroautotrophic nutrition — Heteromeso- 
trophic nutrition- — Heterometatrophic nutrition — Trophic 
specialization — Specific growth factors, or vitamins — 
Other growth factors — Growth stimulants — Growth in 
cultures as a population problem — The initial popula- 
tion — Growth in relation to waste products — Growth in 
relation to food concentration — Growth in relation to 
pH of the medium — Oxygen relationships — The redox 
potential — Growth in relation to temperature — Growth 
in relation to light and darkness — Acclimatization — 
Literature cited 

X. The Growth of the Protozoa By Oscar W. Richards, 

Research Biologist, Spencer Lens Co?npany ... 517 

Methods for the measurement of growth — The growth of 
individual Protozoa — The growth of colonial Protozoa — • 
Pedigree isolation culture and life cycles — Protozoan suc- 
cessions: nonlaboratory — Protozoan successions: labora- 
tory — Autocatalysis and allelocatalysis — Nutrition and 
growth — The growth of populations — The struggle for 
existence — Literature cited 

XI. The Life Cycle of the Protozoa By Charles Atwood 

Kofoid, University of California, Berkeley . . . 565 

Introduction — Asexual reproduction in alternating binary 
and multiple fission (Type I) — Alternation of asexual 
and sexual reproduction (Type II) — The life cycle of 
Eimeria schubergi — The life cycle of Plasmodium 
vivax — ^The life cycle of Paramecium caudatum — Litera- 
ture cited 

XII. Fertilization in Protozoa By John P. Turner, Univer- 
sity of Minnesota 583 

Copulation — Gametic meiosis and fertilization — Autog- 
amy — Zygotic meiosis — Significance of fertilization — 



xii CONTENTS 

Conjugation — The macronucleus during conjugation — 
Conjugant meiosis — Literature cited 

XIII. Endomixis By Lorande Loss Woodruff, Yale Uni- 
versity 646 

Macronuclear reorganization — Endomictic phenomena — ■ 
Autogamy — Periodicity of endomixis — Genetical studies 
on endomixis — Conckisions — Literature cited 

XIV. Sexuality in Unicellular Organisms By T. M. Sonne- 

born, University of Indiana GGG 

Sexuality in Cblamydomonas — The kinds of gametic 
differences observed in Cblamydomonas — The nature of 
the physiological differences between gametes in 
Chlamydomonas — Interpretation of the sexual phe- 
nomena in Chla?77ydomonas — Sexuality in Paramecium 
and other ciliate Protozoa — Sexual differences between 
conjugant individuals — Mating types in relation to the 
Maupasian life cycle — The role of environmental con- 
ditions in determining conjugation — Sex differences be- 
tween gamete nuclei — Significance of the diversities be- 
tween conjugants and between gamete nuclei — Literature 
cited 

XV. Inheritance in Protozoa By H. S. Jennings, Univer- 
sity of California, Los Angeles 710 

Types of reproduction and inheritance — Inheritance in 
uniparental reproduction — Material processes — Inheri- 
tance of characteristics — Changes in inherited characters 
in uniparental reproduction — Age changes, sexual imma- 
turity, and maturity — Inherited degenerative changes re- 
sulting from unfavorable conditions — Inherited acclima- 
tization and immunity — Inherited environmental modifi- 
cations in form and structure — Variation and its inheri- 
tance occurring without obvious action of diverse en- 
vironments — Summary and interpretation — Inheritance 
in biparental reproduction — Biparental inheritance in 
haploids: Flagellata — Sex inheritance and sex-linked in- 
heritance — Biparental inheritance in diploids: Ciliata — 
Inheritance of mating type in Paramecium aurelia — In- 
heritance of mating type in Paramecium bursaria — Effect 
of the cytoplasm and its relation to nuclear constitution — 
Literature cited 



CONTENTS xiii 

XVI. The Protozoa in Connection with Morphogenetic 
Problems By Francis M. Summers, College of the 
City of New York 772 

Physiological regeneration — Some of the factors in re- 
generation — External environment — Cyclical variations 
— Racial variations — Degree of injury and reorganiza- 
tion — The size factor — The nuclei in regeneration — 
Behavior of fragments: grafting and reincorporation — 
Regeneration and division — Polarity changes and proto- 
plasmic streaming — Physiological gradients — Regenera- 
tion in colonial forms — Literature cited 

XVII. Certain Aspects of Pathogenicity of Protozoa By 

Elery R. Becker, lotva State College 818 

Problems of virulence and pathogenicity — Amoebic 
dysentery and bacterial complications — Malaria: Plas- 
modium vivax — Variability in strains and in host re- 
sponse — Coccidiosis in poultry — Nutrition and resistance 
- — Conclusions — Literature cited 

XVIII. The Immunology of the Parasitic Protozoa By Wil- 
liam H. Taliaferro, University of Chicago . . . 830 

The physical bases of immunity — The cells involved in 
immunity — Antibodies and antigens involved in immu- 
nity — The cellular and humoral aspects of immunity — 
Role of immune processes in the development of proto- 
zoan infections — General methods — Malaria — Leishma- 
niasis — Nonlethal infection with the Trypanosoma lewisi 
group of trypanosomes — Continuous fatal trypanosomi- 
asis in the mouse and sometimes in the rat — Intermittent 
fatal trypanosomiasis in various laboratory animals — 
Practical applications of immune reactions — Artificial 
immunization — Immunological reactions used in diagno- 
sis — Immunological reactions in relation to classifica- 
tion — Literature cited 

XIX. Relationships between Certain Protozoa and Other 
Animals By Harold Kirby, Jr., University of Cali- 
fornia, Berkeley 890 

Accidental and facultative parasitism — Systematically 
related free-living and symbiotic Protozoa — Mastigo- 



xiv CONTENTS 

phora — Holotricha — Distributional host relationships 
and host-specificity in representative symbiotic faunules — ■ 
General considerations — Ciliates of sea urchins — Proto- 
zoa of termites and the roach Cryptocercus — Adaptive 
host relationships in morphology and life history — 
General considerations — Thigmotricha — Ptychostomidae 
— Astomata — Conidiophrys — Apostomea — Physiological 
host relationships illustrative of mutualism and com- 
mensalism — Flagellates of termites and Cryptocercus — 
Ciliates of ruminants — Literature cited 

XX. Organisms Living on and in Protozoa By Harold 

Kkby, Jr., University of California, Berkeley . . . 1009 

Epibiotic schizomycetes — Schizomycetes on Mastigo- 
phora — Endobiotic schizomycetes — Associations of a 
constant character — Associations of an occasional char- 
acter- — Sphaerita and Nucleophaga — Historical account 
and distribution — Life history and structure — Effect on 
host — Parasites of the nucleus of Trichonympha — 
Phycomycetes other than Sphaerita and Nucleophaga — 
Protozoa — Phytomastigophora- — Zoomastigophora — Sar- 
codina — Sporozoa — Ciliophora — The genus Amoeho- 
phrya Koeppen — Metazoa — Literature cited 

Index ■ 1115 



•v 



Qd 






/Ji- 



TABLES 

1. Structural components of the fibrillar system of Paramedmn 196 

2. Rate of locomotion of Amoeba proteus in sodium and cal- 

cium salt solutions 338 

3. Sensitivity of respirometers 354 

4. Measurements of protozoan respiration 362 

5. Division rates of Protozoa with constant conditions . . . 528 

6. The effect of hydrogen-ion concentration on the growth of 

Protozoa 540 

7. Breeding relations in Chlamydomonas paradoxa and C. 

pseudoparadoxa 672 

8. Breeding relations in Chlamydomonas sp. [coccifera?), C. 

braimii, C. dresdensis, C. eugametos, and C. pauper a . . 673 

9. Grades of sex reaction in mixtures of sexes G to O from 

the Chlan2ydomonas species C. hraunti, C. dresdensis, and 

C. etigametos 675 

10. System of mating relations in Chlamydomonas hraunil, C. 

dresdensis, and C. eugametos. Observations and inter- 
pretations of Moewus 680 

11. Results of mixing together animals from different cary- 

onides of stock F, Paramecium aurelia 692 

12. The system of breeding relations in Paramecium aurelia . 693 

13. The system of breeding relations in Paramecium hursaria . 695 

14. Environmental modifications, Chlamydomonas debaryana . 722 

15. Early results of selection for low and high numbers of 

spines, Difflugia corona 727 

16. Later results of selection for low and high numbers of 

spines, Difflugia corona 727 

17. Inheritance of spine length, with regression toward the 

mean, Difflugia corona 728 

18. Inheritance of mating types in Paramecium bursaria . . . 759 



xvi TABLES 

19. Mating types of descendant clones, Paramecium bursaria . 760 

20. Lengths in microns of the two races crossed with the re- 

sulting final lengths of the offspring, Paramecium cauda- 

tum 765 

21. Table of minimum volumes necessary for regeneration . . 786 



^ 








ILLUSTRATIONS 

Figures 

1. General morphology of Ur onychia trans juga 16 

2. Uronychia trans juga; merotomy and regeneration ... 17 
3A. Uroleptus mobilis; stages in the fusion of the macronuclei 

prior to cell division 

3B. Uroleptus mohilis; the nuclei in late division stages 

4. Nuclear clefts in the macronuclei of Uroleptus halsey't . 

5. Conchophthirus mytili; extrusion of chromatin, during divi 

sion 

6. Aspidisca lynceus; stages in macronuclear reorganization 

during division 23 

7. Lophomonas blattarum; division and reorganization of the 

nucleus 

8. Chilodonella uncinatus; replacement of pharyngeal basket 

and mouth 

9. Uroleptus mohilis; old-age specimens showing the degenera- 

tion of the macronucleus and the loss of micronuclei . 

10. Uroleptus mohilis; graph representing the life history by 

ten-day intervals 30 

11. Glaucojna [Dallasia) front ata; general morphology of a 

vegetative individual 32 

12. Gametogenesis in Glaucoma (Dallasia) frontata .... 33 

13. Uroleptus mohilis; conjugation and merotomy 34 

14. Uroleptus mohilis; formation of the new macronucleus after 

conjugation 37 

15-16. Mitochondria in Lechriopyla mystax 114 

17. Ichthyophthirius multifiliis; series showing mitochondria and 

the secretion of paraglycogen 114 

18-21. De novo origin of mitochondna. in Monocystis . . . . 114 

22-25. Mitochondtis. in Amoeba proteus 114 

26-27. Mitochondria and protein reserves in Aggregata eberthi 114 

28-29. Mitochondria in conjugants of Bursaria truncatella . . 114 



19 
20 
21 

22 



26 



27 



29 



xviii • ILLUSTRATIONS 

30. The association of mitochondria with the gastriole in Amoeba 

protens 123 

31. The association of the gastrioles and the digestive granules 

in Ichthyophthirms multifiUis 130 

32-35. Morphological variations in the segregation granules of 

Opalhia rcmarum 133 

36-37. Segregation granules in Trypanosomd diemyctylt . . . 134 

38. Stages in the resorption of a segregation granule in Amoeba 

proteus 136 

39. Dictyosomes from Haptophrya mkhiganensis I4l 

40. Dividing dictyosome in Lecudina brasili I4l 

41. Stages in the secretion of neutral fat by Ichthyophthirms 

mnltijiliis I4l 

42-45. Dictyosomes during the life cycle of Lecudina brasili . I4l 
46-47. The effect of centrifuging upon the distribution of cyto- 
plasmic granules l42 

48. Aggregation and disappearance of excretory granules dur- 
ing pulsatory cycle of the contractile vacuole in Ichthy- 

ophthirius multifiliis 146 

49-50. Excretory granules and the contractile vacuole in Poly- 
plastron multivesiculatum 146 

51. Excretory granules associated with the contractile vacuole 

in Dogielella sphaerii 146 

52. "Nephridialplasm" of Cam panel la umbellaria 146 

53-55. Excretory granules in living cihates 149 

56-64. Carbohydrate reserves in various Protozoa 156 

65. The association between glycogen and the parabasal body 

in Crypt obi a helicis 158 

G6. The formation and release of protein granules from the 

m2iCton\ic\eus oi Ichthyophthirius jnultifliis 164 

67-69. External secretion in Euglypha and Ichthyophthirius . l67 

70. Accessory bodies being formed from the neuromotor ring in 

Haptophrya michiganensis 179 

71. Pellicular pattern and longitudinal fibrils connecting basal 

granules in Paramecium 195 

72. Diagram of Gitter (lattice) with attached trichocysts and 

of the neuronemes connecting bases of the cilia . . . . 197 



ILLUSTRATIONS xix 

73. Connecting branch from neuroid to myoneme in Stentor . 203 

74. External fibrillar system of Euplotes patella 207 

75. Stalk of Zoothamnium arbuscula 209 

76. Spasmonem in Zoothamnium 210 

77. Three components of Spironem of ZcoZ/j^i^ww/V/w . . . . 211 

78. Pellicular structure and myonemes in Zoothamn'unn . . . 212 

79. Myonemes of stalk sheath of Vovthella 213 

80. Arrangement of second complex of body fibrils in Epistylis 214 

81. Fibrillar system in Chlamydodon sp 231 

82. Fibrillar system of peristomal region in Conchophthirus 

magna 234 

83. Neuromotor apparatus of D/Ve*/?///! _^iff(^j 235 

84. Neuromotor system in Entodhcus horealis 237 

85. Pellicular fibrils of £;^z/(9r/j//?/(i/V^';;/ ^t/wz/ 239 

86. Optical section of Eupoterion pern'ix 240 

87. Neuromotor system of Haptophrya michiganensis .... 242 

88. Cross section of peripheral region of Balantidium coli . . 245 

89. Diagram of fibrillar system in Nyctotherus hylae .... 249 

90. Spirostomum amhiguum. Diagram of membranelles and 

their intracytoplasmic structures 251 

91. Fibrillar complex of cytostome in Oxytrkha 255 

92. Section through anterior end of Uroleptus halseyi .... 256 

93. Camera sketch of horizontal optical section of Amoeba pro- 

teus 272 

94. Curves showing relation between luminous intensity, reaction 

time, stimulation period, and latent period in Amoeba 

proteus 274 

95. Camera drawings of Amoeba sp. illustrating the response to 

localized illumination 276 

96. Relation between adaptation to light of difi^erent intensities 

and rate of locomotion in Amoeba proteus 278 

97. Camera outlines representing different stages in the process 

of orientation in Amoeba proteus 280 

98. Diagrams showing the position of the flagellum of Euglena 

as seen in a viscid medium 281 

99. Euglena sp. in a crawling state, showing details in the proc- 

ess of orientation 284 



XX ILLUSTRATIONS 

100. Side view of anterior end of Eiiglena viridis 285 

101. Graphs showing the relation between the direction of loco- 

motion of flagellates observed in a field of light produced 
by two horizontal beams crossing at right angles and that 
demanded by the "Resultantengesetz" 288 

102. Curves representing the distribution in the spectrum of stim- 

ulating efficiency 289 

103. Camera drawings illustrating the response of Peranenia to 

contact or to rapid increase in luminous intensity . . . 291 

104. Graph showing the effect of dark-adaptation on sensitivity 

to light in Peranenia trkhophoruiu 292 

105. Graphs showing rate of light-adaptation in Peranenia . . 293 

106. Graphs showing relation between luminous intensity and re- 

action time, exposure period, latent period, and energy 

{for Peranema) 294 

107. Stentor coeruleus in the process of orientation 296 

108. Arrangement of zooids in a colony of Volvox 298 

109. Sketches showing the structure of the eyespot in Volvox and 

its action on light entering the pigment cup at different 
angles 299 

110. Diagrammatic representation of the process of orientation 

in Volvox 301 

111. Sketches illustrating the effect of a galvanic current on a 

monopodal Amoeba moving toward the cathode . . . 307 

112. Sketches illustrating the effect of a galvanic current on mono- 

podal amoebae moving toward the anode in a weak cur- 
rent 308 

113. Graphs showing comparative effect of different densities of 

current on the rate of locomotion 310 

114. A series of camera sketches of an Amoeba, showing the ef- 

fect of an alternating current 312 

115. Progressive cathodic reversal of the cilia and change of form 

in Paramecium as the constant electric current in made 
stronger 322 

116. Paramecium showing reversal in the direction of the stroke 

of the cilia in a galvanic current 325 

117. Sketch showing in a stationary photopositive colony of Vol- 



ILLUSTRATIONS xxi 

vox the effect of a galvanic current on the currents of 
water produced by the flagella 328 

118. Diagrams illustrating the effect of direct current on the dis- 

tribution of ions in colonies of Vol vox and their response 330 

119. The relation between rate of locomotion, gel/sol ratio, and 

hydrogen-ion concentration in a balanced salt solution . 334 

120. The relation between rate of locomotion, gel/sol ratio, 

hydrogen-ion concentration, and sodium-ion concentra- 
tion 335 

121. The relation between rate of locomotion, gel/sol ratio, hydro- 

gen-ion concentration, and calcium-ion concentration . . 336 

122. The effect of adding calcium in different concentrations to 

0.005 N sodium solutions, on the relation between hydro- 
gen-ion concentration, rate of locomotion, and gel/sol 
ratio 337 

123. Capillary tube used for the sterilization of Trichomonas 

hominh 456 

124. Migration tube 459 

125. V migration tube for semisolid media 460 

126. Migration-dilution apparatus drawn to show construction 462 

127. Details of construction of the sponge-and-glass plunger for 

the collection of cysts 465 

128. Growth phases in a hypothetical population 494 

129. Hypothetical modifications of the normal growth of a popu- 

lation, from the initial stationary to the maximal stationary 
phase 496 

130. Growth in length and in area of 'Paramecium caudatum . . 521 

131. Growth in breadth and thickness of Paramecium caudatum 522 

132. Growth in volume of Paramecium., Frontonia, and Hartman- 

ella 524 

133. Hypothetical curves to illustrate phases of population growth 546 

134. Population growth curves of Euglena, Paramecium, Siylo- 

nychia, and Mayorella 549 

135. Diagram of the life cycle of Trichomonas augusta .... 570 

136. Diagram of the life cycle of Endamoeha coli {Councilmania 

lafleuri) 572 



xxii ILLUSTRATIONS 

137. Diagram of the alternating sexual and asexual reproduction 

in the life cycle of Ehiieria schuhergi 574 

138. Dia.gra.m of the life cycle of Plasmodium vwax 577 

139. Diagram of the life cycle of Payau/eci/^m caudatum . . . 579 

140. Copromonas subtilis in hologamous copulation 585 

14 1. Autogamy in Sappinia diploklea 596 

142. Actinophrys sol in autogamous fertilization 599 

143. Progamic divisions in Monocystis rostrata 601 

144. Isogamous gamete formation and fertilization in Ophryo- 

cyst'is mesnili 603 

145. Diagram of the life cycle of Sphaeromyxa sdbrazesi . . . 607 

146. A ipa.ir of Euplotes patella in conjugSLt'ion 619 

147. Diagram of ciliate conjugation 625 

148. Stages in the first maturation division of Euplotes patella . 628 

149. First maturation spindles of Uroleptus mohilis 630 

150. Second meiotic division in Euplotes patella 632 

151. Third maturation division and the fertilization nucleus in 

Euplotes patella 633 

152. Migration of the pronuclei across the protoplasmic bridge 

of conjugating Chilodonella unc'matus 635 

153. Stages in the progress of the reorganization bands through 

the macronucleus of Aspidisca 646 

154. Macronuclear dissolution in Blepharisma undulans . . . 647 

155. General plan of the usual nuclear changes during endomixis 

in Parameciujn auvelia 648 

156. Possible methods of micronuclear and cell division at the cli- 

max of endomixis in Paramecium aurelia 649 

157. Diagram of the normal process of endomixis in Trkhodina 

sp 651 

158. Endomixis in Paramecium multimicronucleatum .... 652 

159. Endomixis in Paraclevelandia simplex 653 

160. Nuclear changes during autogamy in Paramecium aurelia . 655 

161. Hemixis in Paramecium aurelia 656 

162. Clim20!L of endomixis in Paramecium aurelia 657 

163. Axxtogdimy in sewetA tdiCes of Paramecium aurelia . . . . 658 

164. Graph of division rate of Parameciujn aurelia 660 



ILLUSTRATIONS xxiii 

165. Group formation in Chlamydonionas, showing groups 

formed in a mixture of cells differing in sex 674 

166. The mating reaction in Pi^;w;2^r/V/w ^//rj,^;74 . . . . . 691 

167. Conjugation in Cycloposthium hipalmatiim showing hetero- 

morphic pronuclei 702 

168. Dif^ugia corona; members of four different clones, showing 

diversities in characteristics 724 

169. Polytoma uvella and P. pascheri; the four races used in the 

breeding experiments of Moewus 733 

170. Results of a cross between two species of Poly to/// a . . . 736 

171. New combinations resulting from crossing over in Polyto///a 738 

172. Diagram of the sex chromosome of Polyto/zia pascheri plus 

and P. uvella minus 743 

173. Diagram of the sex chromosomes produced by crossing over 

between the chromosomes of Polyto//ia pascheri plus and 

P. uvella minus 744 

174. Diagram of pregamic divisions and pronuclear exchange in 

ciliates 751 

175. The four clones of Para///eciu/// produced from the two ex- 

conjugants of a pair, in the experiments of Sonneborn and 

of Jennings 753 

176. Change of size resulting from conjugation of individuals of 

large and small races of Para///eciu//i caudatu//? .... 762 

177. Changes in mean size of the descendants of the two members 

of an unequal pair of Para///eciu//i caudatu/// .... 763 

178. Different ultimate mean sizes reached by descendants of dif- 

ferent pairs from crosses of the same two races of Para- 

nieciu/// caudatu/// 764 

179. Regeneration in. Uro/2ych/a tra/isfuga 776 

180. Regeneration in Ur onychia uncinata 783 

181. Reincorporation in Difflugia pyrifor/zjis 794 

182. Divisional and physiological reorganization in Uronychia . 797 

183. Diagram showing delayed regeneration in Para/neciu/// cau- 

datum 799 

184. Successive stages in the regulation of the mouth in a piece 

of Spirosto/nu//j with the mouth at the anterior end . . . 803 

185. A relatively mature colony of Zootha/nnium alternans . . 806 



xxiv ILLUSTRATIONS 

186. A seventy-two-hour regenerate produced from a lateral cell 

of the first branch generation in Zoothamnium .... 808 

187. Branch C of a Zoothamnium colony fifty-six hours after in- 

jury to the neuromuscular cord 810 

188. The changes in number of Plasmod'nwi brasilianum and the 

percentage of segmenters during the acute rise and crisis 

of the infection in a Central American monkey . . . 845 

189. The changes in number of Trypanosoma lew} si and the co- 

efficient of variation and percentage of division forms dur- 
ing the course of infection in the rat 856 

190. The demonstration of ablastin against Trypanosoma lewisi 

by passive transfer 859 

191. The changes in number of Trypanosoma rhodesiense and 

the coefficient of variation during the course of infection 

in a mouse 862 

192. The changes in number of Trypanosoma rhodesiense and 

the coefficient of variation during the course of infection 

in a guinea pig 865 

193. The demonstration of a trypanolysin against a passage strain 

of Trypanosoma equinum by passive transfer .... 867 

194. Posterior end of larva of y^^-'i^^j (Stegomyia) scutellaris paisi- 

sitized by ciliates 897 

195. One-day-old nymph of Kalotermes jlavicolUs, receiving proc- 

todaeal food from the female termite 927 

196. Streblomastix strix attached to the lining of the hind-gut of 

Zootermopsis angusticollis 930 

197. Fixation mechanisms in peritrichs 931 

198. Fixation apparatus of Cyclochaeta [Urceolaria) korschelti . 932 

199. Thigmotricha 939 

200. Thigmotricha 941 

201. Ptychostomidae 944 

202. Skeletal structures and attachment organelles in Astomata . 948 

203. Anoplophrya (Collinia) circulans in Asellus aquaticus . . 951 

204. Conidiophrys pilisuctor on Corophium acherusicmn . . . 955 

205. Synophrya hyperirophica; dia-gad-tn of Vde cycle 958 

206. Ingestion of plant material by Ophryoscolecidae .... 978 

207. Ingestion and digestion of starch in Eudiplodinium mediutn 980 



ILLUSTRATIONS xxv 

208. Fusifon?7Js-\ike rods adherent to the surface of flagellates . 1012 

209. Adherent microorganisms on flagellates of termites . . . 1014 

210. Adherent microorganisms on flagellates of termites . . . IOI6 

211. Spirochetes adherent to Stephanonympha sp. from Neo- 

termes insular is 1017 

212. Surface microorganisms on various Protozoa 1020 

213. Bacteria adherent to ciliates 1023 

214. Characteristic bacteria adherent to the pellicle of Cyclidium 

from the intestine of sea urchins 1024 

215. Bacteria [Cladothyix pelomyxae Veley, and a small species) 

in Pelomyxa palustris Greeff 1026 

216. Microorganisms in Stephanonympha and Caduceia . . . 1031 

217. Developmental stages of Sphaerita in several Protozoa . . 1049 

218. Nucleophaga 1055 

219. Microorganisms in Trichonynipha IO6O 

220. Nuclear parasites of Trichonynipha sp. from Procryptoter- 

?nes sp 1062 

221. Filamentous fungi parasitic on Amoeba proteus .... IO66 

222. Small Protozoa ectoparasitic on Chilomonas and Colpoda . 1071 

223. Entamoeba in Zelleriella and Raphidiocystis on Paramecium 1077 

224. Cysts of two species of Amphiacantha, metchnikovellids 

parasitic in the gregarine Ophiodtna elongata . . . . 1081 

225. Opisthonecta henneguyi, parasitized by Endosphaera engel- 

manni 1088 

226. Amoebophrya in Radiolaria 1091 

Plates 

1. Sluggish phagocytosis of Plasmodium brasilianum by macro- 

phages in control of the American monkeys during the 
acute rise of the malarial infection and the concentration of 
P. brasilianum in Billroth cords of the spleen at the initi- 
ation of the crisis 850 

2. Intense phagocytosis of Plasmodium brasilianum by macro- 

phages in Central American monkeys at the height of the 
crisis of the malarial infection and malarial pigment, the 
residue of parasites after the intense phagocytosis, in the 
macrophages shortly thereafter 851 



xxvi ILLUSTRATIONS 

3. Portions of a venous sinus and Billroth cords in the spleen of 

an uninfected rhesus monkey and comparable portions from 
a rhesus monkey during the late acute rise of an infection 
'w'i.ih Plasmodium cynomolgi 852 

4. A nodule in the white pulp of the spleen during lymphoid hy- 

perplasia associated with the late acute rise of Plasmodium, 
cynomolgi in a rhesus monkey 853 



LIST OF ABBREVIATIONS 

Abh. bayer. Akad. Wiss. : Abhandlungen der Kgl. Bayerischen Akademie der 

Wissenschaften. Math.-Phys. Kl. Miinchen. 
Abh. naturw. Ver. Bremen.: Abhandlungen hrsg. vom Naturwissenschaft- 

Hchen Verein zu Bremen. 
Abh. sencken b. natur f . Ges. : Abhandlungen hrsg. von der Senckenbergischen 

Naturforschenden Gesellschaft. Frankfurt a. M. 
Acta Biol. exp. : Acta biologiae experimentalis. Warsaw. 
Act. Zool. Stock: Acta Zoologica: Internationell Tidskrift for Zoologica. 

Stockholm. 
Act. Sci. Indust.: Actualites scientifique et industriel. Paris. 
Allatt. Kozlem. : Allattani Kozlemenyek. Budapest. 
American Academy of Arts and Sciences, see Mem. Amer. Acad. Arts Sci. ; 

Proc. Amer. Acad. Arts Sci. 
Amer. J. Anat. : American Journal of Anatomy. Baltimore. 
Amer. J. Bot. : American Journal of Botany. Lancaster. 
Amer. J. Hyg. : American Journal of Hygiene. Baltimore. 
Amer. J. Path.: American Journal of Pathology. Boston. 
Amer. J. Physiol.: American Journal of Physiology. Boston. 
Amer. J. Psychiat. : American Journal of Psychiatry. Baltimore. 
Amer. J. publ. Hlth. : American Journal of Public Health. New York. 
Amer. J. Syph.: American Journal of Syphilis. St. Louis, Mo. 
Amer. J. trop. Med. : American Journal of Tropical Medicine. Baltimore. 
Amer. Midi. Nat.: American Midland Naturalist. Notre Dame, Ind. 
Amer. mon. micr. J. : American Monthly Microscopical Journal. Washington. 
Amer. Nat.: American Naturalist. Boston. 
Anat. Anz.: Anatomische Anzeiger. Jena. 
Anat. Rec. : Anatomical Record. Philadelphia. 
An. Fac. Med. Porto. : Anais da Faculdade de Medicina do Porto. 
An. Inst. Biol. Univ. Mex.: Anales del Institute de biologia, Universidade de 

Mexico. Mexico. 
Ann. appl. Biol.: Annals of Applied Biology. Cambridge. 
Ann. Bot., Lond.: Annals of Botany. London. 

Ann. Fac. Med. S. Paulo. : Annaes da Faculdade de Medicina de Sao Paulo. 
Ann. Igiene (sper.) : Annali d'igiene (sperimentale) . Torino. 
Ann. Inst, oceanogr. Monaco.: Annales de I'lnstitut oceanographique de 

Monaco. Monaco. 
Ann. Inst. Pasteur.: Annales de I'lnstitut Pasteur. Paris. 
Ann. Mag. Nat. Hist.: Annals and Magazine of Natural History. London. 



xxviii LIST OF ABBREVIATIONS 

Ann. Microg. : Annales de Micrographie spedalement consacree a la bac- 

teriologie au protophytes et aux protozoal res. Paris. 
Ann. Mus. Stor. nat. Genova.: Annali del Museo cirico di storia naturale. 

Geneva. 
Ann. Mus. 200I. polon.: Annales musei zoologici polonici. Warszawa. 
Ann. Natal Mus.: Annals of the Natal Museum. Pietermaritzburg. 
Ann. N. Y. Acad. Sci.: Annals of the New York Academy of Sciences. 

New York. 
Annot. zool. jap.: Annotationes zoological japonenses. Tokyo. 
Ann. Parasit. hum. comp.: Annales de parasitologie humaine et comparee. 

Paris. 
Ann. Physiol. Physicochim. biol. : Annales de physiologie et de physicochimie 

biologique. 
Ann. Protist.: Annales de protistologie. Paris. 
Ann. Sci. nat.: Annales des sciences naturelles. (a) Botanique. (b) Zoologie. 

Paris. 
Ann. Soc. beige Micr. : Annales de la Societe beige de microscopie. Bruxelles. 
Ann. Soc. Sci. med. nat. Brux. : Annales (et Bulletin). Societe R. des sciences 

medicales et naturelles de Bruxelles. 
Ann. trop. Med. Parasit.: Annals of Tropical Medicine and Parasitology. 

Liverpool. 
Anz. Akad. Wiss., Wien.: Anzeiger der Kaiserlichen Akademie der Wissen- 

schaften. Math. Kl. Wien. 
Arb. Gesundh. Amt. Berl. : Arbeiten aus dem Kais. Gesundheitsamte. Berlin. 
Arb. Staatsinst. exp. Ther. Frankfurt.: Arbeiten aus dem Staatsinstitut fiir 

experimentelle Therapie und dem Georg. Speyer-Hause zu Frankfurt a. M. 

Jena. 
Arb. zool. Inst. Univ. Wien.: Arbeiten aus den Zoologischen Institut der 

Univ. Wien u. der Zoolog. Station in Triest. Wien. 
Arch. Anat. micr.: Archives d'anatomie microscopique. Paris. 
Arch. Anat. Physiol. Lpz.: Archiv fur Anatomie und Physiologie. Leipzig. 
Arch. Anat. Physiol, wiss. Med.: Archiv fiir Anatomie, Physiologie und 

wissenschaftliche Medicin. Leipzig. 
Arch, argent. Enferm. Apar. dig.: Archives argentinos de enfernedades del 

aparato digestive de la nutricion. Buenos Aires. 
Arch. Biol. Paris.: Archives de biolegie. Paris. 

Arch. exp. Zellforsch.: Archiv fiir experimentelle Zellferschung. Jena. 
Arch. Hyg. Berl.: Archiv fiir Hygiene (und Bakteriologie) . Miinchen u. 

Berlin. 
Arch. Inst. Pasteur Afr. N. : Archives des Instituts Pasteur de I'Afrique du 

Nerd. Tunis. 
Arch. Inst. Pasteur Alger.: Archives de I'lnstitut Pasteur d'Algerie. Alger. 
Arch, intern. Med.: Archives of Internal Medicine. Chicago. 
Arch. int. Physiol. : Archives internationales de physiologie. Liege et Paris. 



LIST OF ABBREVIATIONS xxix 

Arch. Med. exp. : Archives de medecine experimentale et d'anatomie patholo- 

gique. Paris. 
Arch. mikr. Anat. : Archiv fiir Anatomic (und Entwicklungsmechanik) . 

Bonn. 
Arch. Mikrobiol. : Archiv fiir Mikrobiologie. Berlin. 
Arch. Naturgesch. : Archiv fiir Naturgeschichte. Berhn. 
Arch, parasit. : Archives de parasitologic. Paris. 
Arch. Path. Lab. Med.: Archives of Pathology and Laboratory Medicine. 

Chicago. 
Arch, physiol. norm. path. : Archives de physiologic normalc et pathologique. 

Paris. 
Arch. Protistenk. : Archiv fiir Protistenkunde. Jena. 

Arch. russ. d'Anat. : Archives russes d'anatomie, d'histologie et d'embryologie. 
Arch. russ. protist. : Archives russes de protistologie. 
Arch. Schiffs- u. Tropenhyg. : Archiv fiir Schiffs- u. Tropenhygiene. . . . 

Leipzig. 
Arch. Sci. biol., St. Petersb. : Archives des sciences biologiques. St. Peters- 

bourg. 
Arch. Sci. phys. nat. : Archives des sciences physiques et naturelles. Geneve, 

Lausanne, Paris. 
Arch. Tierernahr. Tierz. : Archiv fiir Tiererniihrung und Tierzucht. Berlin. 
Arch. wiss. prakt. Tierheilk. : Archiv fiir wissenschaftliche u. praktische Tier- 

heilkunde. Berlin. 
Arch. Zellforsch. : Archiv fiir Zellforschung. Leipzig. 

Arch. zool. exp. gen.: Archives de zoologie experimentale et generale. Paris. 
Arch. zool. (ital.) : Archivio zoologico italiano. Napoli. Torino. 
Arhiva vet. : Arhiva Veterinara. Bucuresti. 
Arsskr. Lunds Univ.: Arsskrift Lunds Universitet. 
Atti Accad. gioenia.: Atti della R. Accademia gioenia di scienze naturali. 

Catania. 
Atti della R. Accademia dei Lincei, see R. C. Acad. Lincei. 
Atti Soc. Studi Malar.: Atti della Societa per gli studi della malaria. Roma. 
Aust. J. exp. Biol. med. Sci. : Australian Journal of Experimental Biology and 

Medical Science. Adelaide. 
Bact. Rev.: Bacteriological Review. Baltimore, Md. 
Beih. bot. Zbl.: Beihefte zum Botanischen Zentralblatt. Cassel. 
Beitr. allg. Bot.: Beitriige zur allgemeinen Botanik. Berlin. 
Beitr. Biol. Pfl.: Beitrage zur Biologic der Pflanzcn. Breslau. 
Ber. dtsch. bot. Ges.: Bericht der Deutschen Botanischen Gesellschaft. Berlin. 
Ber. Ges. Wiss. : Berichte iiber die Verhandlungen der kgl. siichsischen Gesell- 
schaft der Wissenschaften. 
Berl. klin. Wschr. : Berliner klinische Wochenschrift. Berlin. 
Ber. naturf. Ges. Freiburg i. B.: Berichte der Naturforschenden Gesellschaft 

2u Freiburg i. Br. 



XXX LIST OF ABBREVIATIONS 

Ber. senckenb. Ges.: Bericht der Senckenbergischen Naturforschenden Gesell- 
schaft in Frankfurt a. M. Frankfort a. M. 

Ber. wiss. Biol. : Bericht iiber die wissenschaftliche Biologie. Berlin. 

Bibliogr. genet.: Bibliographia genetica. 'sGravenhage. 

Bibliogr. zool.: Bibliographia zoologica. Lipsiae. 

Biederm. Zbl.: Biedermanns Zentralblatt fiir Agrikultur-chemie und ratio- 
nellen Landwirtschaftsbetrieb. Leipzig. 

Bio-chem. J. : Bio-chemical Journal. Liverpool. 

Biochem. Z. : Biochemische Zeitschrift. Berlin. 

Biodyn. : Biodynamica. Normandy, Mo. 

Biol. Bull. Wood's Hole: Biological Bulletin of the Marine Biological Labora- 
tory, Wood's Hole, Mass. 

Biol. Listy. : Biologicke Listy. Prague. 

Biol. Monogr. : Biological Monographs and Manuals. Edinburgh. 

Biologe: Biologe; Monatsschrift zur Wahrung der Belange der deutschen 
Biologen. Munich. 

Biol. Rev.: Biological Reviews and Biological Proceedings of the Cambridge 
Philosophical Society. Cambridge. 

Biol. Zbl.: Biologisches Zentralblatt. Leipzig. 

Biometrika. : Biometrika. Cambridge. 

Bol. biol. Fac. Med. S. Paulo. : Boletim biologico, Laboratorio de parasitologia. 
Faculdade de medicina de Sao Paulo. 

Boll. Lab. Zool. agr. Bachic. Milano. : Bollettino del Laboratorio di zoologia 
agraria e bachicoltura del R. Istituto superiore agrario di Milano. 

Boll. Soc. eustach.: Bollettina della Societa Eustachiana. Camerino. 

Botaniste. : Le Botaniste. Caen, Poitiers, Paris. 

Bot. Gaz. : Botanical Gazette. Chicago. 

Bot. Rev.: Botanical Review. Lancaster, Pa. 

Bot. Ztg. : Botanische Zeitung. Berlin and Leipzig. 

Brazil-med.: Brazil-medico. Rio de Janeiro. 

Brit. J. exp. Biol. : British Journal of Experimental Biology. Edinburgh. 

Brit. J. exp. Path.: British Journal of Experimental Pathology. London. 

Bronn's Klassen.: Bronn's Klassen und Ordnungen des Tierreichs. Leipzig. 

Bull. Acad. Med. Paris.: Academic de Medecine, Bulletin. Paris. 

Bull. Acad. Sci. St.-Petersbourg. : Bulletin de I'Academie Imperiale des sciences 
de St.-Petersbourg. 

Bull. Acad. Sci. U.R.S.S.: Bulletin de I'Academie du sciences de I'U.R.S.S. 

Bull. Amer. Mus. Nat. Hist. : Bulletin of the American Museum of Natural 
History. New York. 

Bull. Bingham oceanogr. Coll.: Bulletin of the Bingham Oceanographic Col- 
lection, Yale University, New Haven. 

Bull. biol. exp. med. U.R.S.S.: Bulletin de Biologique et de Medicine Ex- 
perimentale de L'U.S.S.R. Moscow. 

Bull, biol.: Bulletin biologique de la France et de la Belgique. Paris. 



LIST OF ABBREVIATIONS xxxi 

Bull. Inst, oceanogr. Monaco: Bulletin de I'lnstitut oceanographique de 
Monaco. 

Bull. Inst. Pasteur.: Bulletin de I'lnstitut Pasteur. Paris. 

Bull. int. Acad. Cracovie. : Bulletin international de I'Academie des sciences 
de Cracovie (de TAcadcmie polonaise des sciences). 

Bull. int. Acad. Prag. Sci. Math. Nat. : Bulletin International. Ceski akademie 
ved a umeni v Praze. Sciences, mathematiques et naturelles. Prague. ((!eska 
akademie cesare Frantiska Josef a pro vedy, slovesnost a umeni v Praze.) 

Bull. Mus. Hist. nat. Belg. : Bulletin du Musee Royal d'histoire naturelle de 
Belgique. Bruxelles. 

Bull. N. J. agric. Exp. Stas.: Bulletin of the New Jersey Agricultural Experi- 
mental Stations. New Brunswick. 

Bull. sci. Fr. Belg.: Bulletin scientifique de la France et de la Belgique. 
Londres, Paris, Berlin. 

Bull. Scripps Instn. Oceanogr. tech.: Bulletin Scripps Institution of Oceanog- 
raphy. Technical Series. La Jolla, Calif. 

Bull. Sleep. Sickn. Bur.: Bulletin. Royal Society Sleeping Sickness Bureau. 
London. 

Bull. Soc. hot. Fr. : Bulletin. Societe botanique de France. Paris., 

Bull. Soc. Hist. nat. Afr. N. : Bulletin de la Societe d'histoire naturelle de 
I'Afrique du Nord. Alger. 

Bull. Soc. imp. Natur., Moscow: Societe imperiale des naturalistes. Bulletin. 
Moscow. 

Bull. Soc. med.-chir. Indochine.: Bulletin de la Societe medico-chiruricale de 
rindochine. Hanoi. 

Bull. Soc. Path, exot.: Bulletin de la Societe de pathologie exotique. Paris. 

Bull. Soc. zool. Fr. : Bulletin de la Societe zoologique de France. Paris. 

Bull. U. S. Bur. Fish.: Bulletin of the Bureau of Fisheries. Washington. 

Bull. U. S. nat. Mus.: Bulletin. United States National Museum. Smith- 
sonian Institution. Washington. 

Carnegie Institution, see Pap. Tortugas Lab. ; Publ. Carneg. Instn. ; Yearb. 
Carneg. Instn. 

Camb. Phil. Soc. Proc. : Proceedings of the Cambridge Philosophical Society. 
Cambridge. 

Cellule.: La Cellule. Recueil de cytologic et d'histologie generale. Louvain. 

Cellulose-Chem. : Cellulose-Chemie. (Papierfabrikant. Suppl.) Berlin. 

Chem. Abstr. : Chemical Abstracts. New York. 

Clin. vet. Milano.: La clinica veterinaria. Milano. 

Cold Spr. Harb. Monogr. : Cold Spring Harbor Monographs. Brooklyn, N.Y. 

Cold Spring Harbor Symp. Quant. Biol.: Cold Spring Harbor Symposium on 
Quantitative Biology, 1933. 

Coll. Net.: Collecting Net. Woods Hole, Mass. 

Contr. zool. Lab. Univ. Pa.: Contributions from the Zoological Laboratory, 
Univ. of Pennsylvania. Philadelphia. 



xxxii LIST OF ABBREVIATIONS 

Copeia.; Copeia. Published to advance the science of cold-blooded Verte- 
brates. New York. 

C. R. Acad. Sci. Paris.: Compte rendu hebdomadaire des seances de TAcad- 
emie des sciences. Paris. 

C. R. Acad. Sci. U.R.S.S. : Compte rendu de I'Academie des sciences de 

ru.R.s.s. 

C. R. Ass. Anat.: Compte rendu de I'Association des anatomistes. Paris and 

Nancy. 
C. R. Lab. Carlsberg. : Compte rendu des travaux du Laboratoire de Carls- 
berg. Copenhague. 
C. R. XIL Cong. Int. Zool. : Comptes rendues XIP Congres international de 

la Zoologie. Lisbon, 1935. 
C. R. Soc. Biol. Paris.: Compte rendu hebdomadaire des seances et memoires 

de la societe de biologie. Paris. 
Cytologia, Tokyo.: Cytologia. Tokyo. 
Denkschr. Akad. Wiss. Wien. : Denkschriften der Kaiserlichen Akademie der 

Wissenschaften. Math.-nat. Kl. Wien. 
Denkschr. med.-naturw. Ges. Jena.: Denkschriften der Medizinisch-natur- 

wissenschaftlichen Gesellschaft zu Jena. 
Dtsch. med. Wschr. : Deutsche medizinische Wochenschrift. Leipzig. 
Dtsch. tierarzlt. Wschr.: Deutsche tierarztliche Wochenschrift. Hannover. 
Dutch East Indies Volkksgesondheid.: Dutch East Indies. Dienst der Volks- 

gesondheid in Nederlandsch-Indie. Mededeelingen. 
Ecol. Monogr. : Ecological Monographs. Durham, N.C. 
Ecology.: Ecology. Brooklyn. 
Edinb. New phil. J.: Edinburgh New Philosophical Journal, 1826, 1854, 

1855-'64. 
Ergebn. Biol.: Ergebnisse der Biologie. Berlin. 
Ergebn. Hyg. Bakt. : Ergebnisse der Hygiene, Bakteriologie, Immunitats- 

forschung u. experimentellen Therapie. Berlin. 
Ergebn. inn. Med. Kinderheilk.: Ergebnisse der inneren Medizin u. Kinder- 

heilkunde. Berlin. 
Ergebn. Physiol. : Ergebnisse der Physiologie. Wiesbaden. 
Ergeb. Zool.: Ergebnisse u. Fortschritte der Zoologie. Jena. 
Fauna u. Flora Neapel.: Fauna u. Flora des Golfes von Neapel u. d. angrenz. 

Meeresabschnitte. Berlin. 
Flora, Jena. : Flora, oder allgemeine botanische Zeitung. Jena, Regensburg. 
Folia haemat.: Folia haematologica. Leipzig. 
ForschBer. biol. Sta. Plon.: Forschungsberichte aus der Biologischen Station 

2u Plon. Stuttgart. 
Gegenbaurs Jb.: Gegenbaurs morphologisches Jahrbuch. Leipzig. 
Genetics.: Genetics: a Periodical Record of Investigations bearing on 

Heredity and Variation. Menasha, Wis. 



LIST OF ABBREVIATIONS xxxiii 

Gottingen, mathem. -physical. Kl.: Nachrichten von der Konigl. Gesellschaft 

der Wissenschaften 2u Gottingen. Mathematisch-physikalische Klasse. 

Gottingen. 
Grece med.: Grece medicale. Syra. 
Growth.: Growth. (A journal.) Series of numbered fasciculi at irregular 

intervals. 
Handb. Vererbungsw.: Handbuch der Vererbungswissenschaft. Berlin, 
llinois biol. Monogr. : Illinois Biological Monographs. Univ. of Illinois. 

Urbana. 
Indian J. med. Res.: Indian Journal of Medical Research. Calcutta, 
ndian med. Gaz. : Indian Medical Gazette. Calcutta, 
ndian med. Res. Mem.: Indian Medical Research Memoirs. Calcutta, 
ndust. Engng. Chem. : Industrial and Engineering Chemistry. Easton, Pa. 
nt. Rev. Hydrobiol.: Internationale Revue der gesamten Hydrobiologie u. 

Hydrographie. Leipzig. 
Iowa St. Coll. J. Sci. : Iowa State College Journal of Science. Ames. 

Acad. nat. Sci. Philad.: Journal of the Academy of Natural Sciences of 

Philadelphia. 

agric. Res.: Journal of Agricultural Research. Washington. 

Amer. med. Ass.: Journal of the American Medical Association. Chicago. 

Amer. statist. Ass. : Journal of the American Statistical Association. Boston. 

anat. Paris.: Journal de I'anatomie et de la physiologie normales et 

pathologique de I'homme et des animaux. Paris. 

Anim. Behav. : Journal of Animal Behaviour. Boston. 

appl. Physics: Journal of Applied Physics. Menasha, Wis. 

Bact. : Journal of Bacteriology. Baltimore. 

biol. Chem.: Journal of Biological Chemistry. Baltimore. 

cell. comp. Physiol.: Journal of Cellular and Comparative Physiology. 

Philadelphia. 

Coll. Sci. Tokyo.: Journal of the College of Science, Imp. University of 

Tokyo. 

comp. Neurol.: Journal of Comparative Neurology (and Psychology). 

Philadelphia. 

comp. Path.: Journal of Comparative Pathology and Therapeutics. Edin- 
burgh, London. 

Coun. sci. industr. Res. Aust. : Journal of the Council for Scientific and 

Industrial Research, Australia. Melbourne. 

Dairy Sci.: Journal of Dairy Science. Baltimore. 

Dep. Sci. Calcutta Univ.: Journal of the Department of Science of Cal 

cutta University. Calcutta. 

Elisha Mitchell sci. Soc. : Journal of the Elisha Mitchell Scientific Society, 

Chapel Hill, N.C. 

exp. Biol.: Journal of Experimental Biology. Cambridge. 

exp. Med. : Journal of Experimental Medicine. New York. 



xxxiv LIST OF ABBREVIATIONS 

. exp. Zool.: The Journal of Experimental Zoology. Philadelphia. 

. Fac. Sci. Tokyo Univ.: Journal of the Faculty of Science, Tokyo Imperial 

University. Tokyo. (4) Zool. 
. Genet.: Journal of Genetics. Cambridge. 
. gen. Physiol.: Journal of General Physiology. Baltimore. 
. Helminth.: Journal of Helminthology. London. 
. Immunol.: Journal of Immunology. Baltimore. 
. industr. Engng. Chem.: Journal of Industrial and Engineering Chemistry. 

Easton, Pa. 
. infect. Dis.: Journal of Infectious Diseases. Chicago. 
. Lab. clin. Med. : Journal of Laboratory and Clinical Medicine. St. Louis, Mo. 
. linn. Soc. (Zool.) : Journal of the Linnean Society. (Zoology.) London. 
. Malar. Inst. India.: Malaria Institute of India, Journal. 
. Mar. biol. Ass. U. K. : Journal of the Marine Biological Association 

of the United Kingdom. Plymouth. 
. med. Res.: Journal of Medical Research. Boston. 
. Microbiol. : Journal de microbiologie. Petrograd. Moscou. 
. Morph. : Journal of Morphology (and Physiology) . Philadelphia, Boston. 
. nerv. ment. Dis. : Journal of Nervous and Mental Diseases. New York. 
. Parasit.: Journal of Parasitology. Urbana, 111. 
. Path. Bact. : Journal of Pathology and Bacteriology. London. 
. phys. Chem. : Journal of Physical Chemistry. Ithaca, N.Y. 
. Physiol.: Journal of Physiology. London and Cambridge. 
. Physiol. Path, gen.: Journal de physiologic et de pathologie generale. Paris. 
. prev. Med. Lond. : Journal of Preventive Medicine. London. 
. prev. Med., Oshkosh, Wis.: Journal of Preventive Medicine. Oshkosh, 

Wis. 
. R. Army med. Cps. : Journal of the Royal Army Medical Corps. London. 
. R. micr. Soc: Journal of the R. Microscopical Society. London. 
. Sci. Hiroshima Univ. Journal of Science of the Hiroshima University. 

Hiroshima, Japan. 
. trop. Med. (Hyg.) : Journal of Tropical Medicine (and Hygiene). London, 
ap. J. Zool.: Japanese Journal of Zoology. Tokyo, 
ber. Fortschr. Anat. EntwGesch.: Jahresberichte iiber die Fortschitte der Ana- 

tomie und Entwicklungsgeschichte. Jena, 
b. wiss. Bot. : Jahrbuch fiir wissenschaftliche Botanik. Berlin, 
ena. Z. Naturw. : Jenaische Zeitschrif t fiir Naturwissenschaft. Jena, 
ena. Z. Naturgesch.: Jenaische Zeitschrift fiir Naturgeschichte. Jena. 
Kiev, obshch, estest. Zap.: Kievskve obshchestvo estestvoispytatelei Zapiski. 

Kief. 
Kryptogamenfl. Mark Brandenb.: Kryptogamenflora der Mark Brandenburg 

und angrenzender Gebiete. (Botanische Verein der Provinz Brandenburg) . 

Leipzig. 
Matemat. termesz. estesito.: Matematikai es termeszettudomanyi estesito. 



LIST OF ABBREVIATIONS xxxv 

Math, naturw. Ber. Ung. : Mathematische und naturwissenschaftliche Berichte 

aus Ungarn. [Berlin: Budapest:] Leipzig. 
Math.-nat. KL, Heidelb. Akad. Wiss.: Mathematisch-naturwissenschaftliche 

Klasse, B. Biologische wissenschaften. Heidelberger Akademie der Wissen- 

schaften. 
Medicine.: Medicine. Baltimore, Md. 

Med. Paises calidos. Medicina de los paises calidos. Madrid. 
Mem. Acad. roy. Belg. : Academie royale des sciences de Belgique, Memoires. 

Brussels. 
Mem. Acad. Sci., Paris.: Memoires de 1' Academie des sciences de I'lnstitut 

de France. Paris. 
Mem. Amer. Acad. Arts Sci.: Memoirs of the American Academy of Arts 

and Sciences. Boston. 
Mem. CI. Sci. Acad, polon.: Memoires de la classe des sciences mathematiques 

e*: naturelles. Academie Polonaise des Sciences et des Lettres. Cracovie. 
Mem. Coll. Sci. Kyoto.: Memoirs of the College of Science, Kyoto Imp. Uni- 
versity. Kyoto. 
Mem. Inst. nat. genev. : Memoires de I'lnstitut national genevois. Geneve. 
Mem. Inst. Osw. Cruz.: Memorias do Institute Oswaldo Cruz. Rio de Janeiro. 
Mem. Mus. Hist. nat. Belg.: Memoires du Musee royal d'histoire naturelle 

de Belgique. Bruxelles. 
Mem. Soc. Phys. Geneve.: Memoires de la Societe de physique et d'histoire 

naturelle de Geneve. 
Mem. Soc. zool. tehee. Prague.: Ceskoslovenska spolecnost zoologicka (Soci- 

etas Zoologica cechoslovenica) . Prague. 
Microgr. prep.: Micrographe preparateur; journal de micrograprie generale, 

de technique micrographique et revue des journaux frangais et etrangers. 

Paris. 
Microscope.: Microscope. Ann Arbor; Detroit; Washington. 
Midi. Nat.: Midland Naturalist (Midland union of natural history societies). 

London ; Birmingham. 
Midi. Nat. : Midland Naturalist. Notre Dame, Ind. 
Mikrokosmos. : Mikrokosmos (Deutsche mikrologische Gesellschaft, . . .) 

Stuttgart. 
Mitt. zool. Inst. Univ. Miinster. : Mitteilungen aus dem Zoologischen Institut 

der Westfalischen Wilhelms-Universitat zu Miinster i. W. 
Mitt. Zool. Sta. Neapel.: Mitteilungen aus der Zoologische Station zu 

Neapel. 
Monatsber. preuss. Akad. Wissensch.: Monatsberichte K. preussische Akad- 
emie der Wissenschaften zu Berlin. 
Monit. zool. ital.: Monitore zoologico italiano. Sienna; Firenze. 
Monogr. Inst. Pasteur.: Monographies de I'lnstitut Pasteur. Paris. 
Morph. Jb. : Morphologisches jahrbuch. Leipzig. 
Nature.: Nature. London. 



xxxvi LIST OF ABBREVIATIONS 

Naturwissenschaften. : Naturwissenschaften. Berlin. 

Nova Acta Leop. Carol.: Nova Acta Leopoldinisch-Carolinische deutsche 

Akademie der Naturforscher. Jena. 
Occ. Pubis. Amer. Ass. Adv. Sci.: Occasional Publications, American Associ- 
ation for the Advancement of Science. 
Ohio J. Sci.: Ohio Journal of Science (Ohio State University . . . Ohio 

Academy of Science) . Columbus. 
Ost. bot. Z. : Oesterreichische botanische Zeitschrift. Wien. 
Pap. Tortugas Lab.: Papers from the Tortugas Laboratory (Department of 

Marine Biology) of the Carnegie Institution of Washington. Washington. 
Parasitology.: Parasitology, a supplement to the Journal of Hygiene. Cam- 
bridge. 
Pasteur Institute, see Ann. Inst. Pasteur; Arch. Inst. Pasteur Afr. N. ; Arch. 

Inst. Pasteur Alger. ; Bull. Inst. Pasteur ; Monogr. Inst. Pasteur. 
Pfliig. Arch. ges. Physiol.: Pfliigers Archiv fUr die gesamte Physiologie d. 

Menschen u. d. Tiere. Bonn. 
Philipp. J. Sci.: Philippine Journal of Science. Manila. 
Philos. Trans.: Philosophical Transactions of the Royal Society. London. 
Physics.: Physics. Minneapolis. 
Physiol. Rev.: Physiological Reviews. Baltimore. 
Physiol, russe.: Physiologiste russe. Moscow. 
Physiol. Zool.: Physiological Zoology. Chicago. 

Planta: Planta. (Archiv flir wissenschaftliche Botanik. Berlin. Abt. E.) 
Poult. Sci.: Poultry Science. Ithaca, N.Y. 
Proc. Acad. nat. Sci., Philad.: Proceedings of the Academy of Natural 

Sciences of Philadelphia. 
Proc. Amer. Acad. Arts Sci.: Proceedings of the Academy of American Arts 

and Sciences. Boston. 
Proc. Amer. phil. Soc. Proceedings of the American Philosophical Society. 

Philadelphia. 
Proc. Boston Soc. nat. Hist.: Proceedings of the Boston Society of Natural 

History. Boston. 
Proc. Calif. Acad. Sci.: Proceedings of the California Academy of Sciences. 

San Francisco. 
Proc. Davenport Acad Sci.: Proceedings of the Davenport Academy of 

(Natural) Sciences. Davenport, Iowa. 
Proc. Acad. Sci. Amst. : Proceedings of the Royal Academy of Sciences, 

Amsterdam. 
Proc. Indian Acad. Sci.: Proceedings of the Indian Academy of Sciences. 

Bangalore. 
Proc. K. Akad. Wetensch.: K. Akademie van Wetenschappen, Amsterdam. 

Proceedings of the Section of Sciences (translated). 
Proc. Linn. Soc. N.S.W. : Proceedings of the Linnean Society of New South 

Wales. Sydney. 



LIST OF ABBREVIATIONS xxxvii 

Proc. Minn. Acad. Sci.: Proceedings of tiie Minnesota Academy of Sciences. 
Minneapolis. 

Proc. nat. Acad. Sci., Wash.: Proceedings of the National Academy of Sci- 
ences. Washington. 

Proc. Pa. Acad. Sci.: Proceedings of the Pennsylvania Academy of Science. 
Harrisburg, Pa. 

Proc. R. Irish Acad.: Proceedings of the Royal Irish Academy. Dublin. 
London. 

Proc. roy. Soc. : Proceedings of the Royal Society. London. 

Proc. roy. Soc. Edinb. : Proceedings of the Royal Society of Edinburgh. 

Proc. R. Soc. Med. : Proceedings of the R. Society of Medicine. London. 

Proc. Soc. exp. Biol. N.Y.: Proceedings of the Society for Experimental 
Biology and Medicine. New York. 

Proc. zool. Soc. Lond.: Proceedings of the General Meetings for Scientific 
Business of the Zoological Society of London. 

Protoplasma. : Protoplasma. Leipzig. 

Protop.-Monog. : Protoplasma-Monographien. Berlin, 1928 + 

Protozoology. : Protozoology, a Supplement to the Journal of Helminthology. 
London. 

Publ. Carneg. Instn.: Publications. Carnegie Institution of Washington. 
Washington. 

Publ. Fac. Sci. Univ. Charles.: Spisy Vydavane Pfirodovedeckou Fakultou 
Karlovy University. Praha. 

Publ. Hlth. Rep. Wash. : Public Health Reports. Washington. 

Quart. J. exp. Physiol.: Quarterly Journal of Experimental Physiology. 
London. 

Quart. J. micr. Sci. : Quarterly Journal of microscopical Science. London. 

Quart. Rev. Biol.: Quarterly Review of Biology. Baltimore. 

Radiology.: Radiology. St. Paul. 

Rasseg. faunist. Roma.: Ressegna faunistica. Roma. 

R. C. Accad. Lincei.: Atti della R. Accademia dei Lincei. Rendiconti. CI. di 
sci. fis. mat. e nat. Roma. 

Rec. Malar. Surv. India.: Records of the Malaria Survey of India. Calcutta. 

Rec. zool. Suisse.: Recueil zool. Suisse. Geneva. 

Rep. Brit. Assoc. Adv. Sci.: Report of the British Association for the Ad- 
vancement of Science. Bath. 

Rep. N. Y. St. Conserv. Comm. Report of the New York State Conservation 
Commission. Albany. 

Rep. Sleep. Sickn. Comm. roy. Soc: Report of the Sleeping Sickness Com- 
mission. Royal Society. London. 

Rep. vet. Res. S. Afr. : Report on (of Director of) Veterinary Research. 
Department of Agriculture, Union of South Africa. 

Rev. biol. Nord Fr. : Revue biologique du Nord de la France. Lille. 



xxxviii LIST OF ABBREVIATIONS 

Rev. Microbiol. Saratov.: Revue de microbiologie (et) d 'epidemiologic, (et 

de parasitologic). Saratov. 
Rev. sci. Instrum. : Review of Scientific Instruments. (Optical Society of 

America) Rochester, N.Y. 
Rev. Soc. argent. Biol. : Revista de la Sociedad argentina de biologia. Buenos 

Aires. 
Rev. Suisse ZooL: Revue Suisse de zoologie et Annales de la Societe zoolo- 

gique Suisse et du Museum d'histoire naturelle de Geneve. Geneva, 1893 + 
Rif. med.: Riforma medica. Napoli. 

Riv. Fis. mat. Sci. nat. : Rivista di fisica, matematica e scienze naturali. Pavia. 
Riv. Malariol.: Rivista di malariologia. Roma. 
Roux Arch. EntwMech. Organ.: Wilhelm Roux Archiv fiir Entwicklungs- 

mechamk der Organismen. Leipzig. (Abt. D, Zeitschrift fiir wissenschaft- 

liche Biologie, Berlin.) 
Russ. J. Zool.: Zoologicheskii viestnik. Journal Russe de zoologie. Petrograd. 
Russk. zool. Zh. : Russkii zoologicheskii zhurnal. Revue zoologique russe. 

Moscow. 
S. Afr. J. Sci. : South African Journal of Science. Capetown. 
S. B. Akad. Wiss. Wien.: Sitzungsberichte der Kais. Akademie der Wissen- 

schaften in Wien. 
S. B. bohm. Ges. Wiss.: Sitzungsberichte der Kgl. Bohmischen Gesellschaft 

der Wissenschaften. Prag.-Math.-Nat. Klasse. 
S. B. Ges. Morph. Physiol. : Sitzungsberichte der Gesellschaft fiir Morphologic 

und Physiologic in Miinchen. Miinchen. 
S. B. Ges. naturf . Fr. Bcrl. : Sitzungeberichte der Gesellschaft Naturforschen- 

der Freunde zu Berlin. 
S. B. preuss. Akad. Wiss.: Sitzungsberichte der Kgl. Preussischcn Akademie 

der Wissenschaften zu Berlin. 
Science. Science. New York. 

Sci. Prog.: Science Progress; a quarterly review of current scientific investiga- 
tion. London, 1894-98; Sci. Prog. Twent. Cent. Science Progress in the 

Twentieth Century, a quarterly journal of scientific thought. London, 

1906. 
Scientia, Bologna. : Scientia, rivista di scienza. Bologna. 
Scientia, Ser. biol., Paris.: Scientia. Serie biologique. Paris. 99-04 
Sci. Mem. Offrs. med. san. Dept. Gov. India.: Scientific Memoirs by Officers 

of the Medical and Sanitary Department of the Government of India. 

Calcutta. 
Sci. Mon., Lond.: Scientific Monthly. London. 
Sci. Mon., NY.: Scientific Monthly. New York. 
Sci. Proc. R. Dublin Soc: Scientific Proceedings of the Royal Dublin Society. 

Dublin. 
Sci. Rep. Tohoku Univ.: Science Reports of the Tohoku Imp. University. 

Sendai. S.4, Biology. Sendai. 



LIST OF ABBREVIATIONS xxxix 

Sci. Rep. Tokyo Bunrika Daig. : Science Reports of the Tokyo Bunrika 

Daigaku. Tokyo. 
Scripta bot. PetropoL: Scripta botanica. Leningrad. (Glavnyi botanicheskii 

sad) . 
Smithsonian Institution, see Smithson. misc. Coll. ; Bull. U. S. nat. Mas. 
Smithson. misc. Coll.: Smithsonian Miscellaneous Collections. Washington. 
Soc. geol. France.: Societe geologique de France. Paris. 
Special Rep. Ser. med. res. Counc. : Special report series, Medical research 

council. London. 
Stain Tech.: Stain Technology. Geneva, N.Y. 
Stat. Rep. N.J.: New Jersey. Statistical Report. Trenton, N.J. 
Stazione Zoologica. Naples.: Fauna und Flora des Golfes von Neapel. 1880 + 
Sth. med. J., Nashville.: Southern Medical Journal. Nashville, Tenn. 
Studies Inst. Divi Thomae.: Studies of the Institutum Divi Thomae, Cin- 
cinnati, Ohio. 
Tabl. zool.: Tablettes zoologiques. Poitiers. 

Tijdschr. ned.: Nederlandsch Tijdschrift voor Geneeskunde. Amsterdam. 
Trab. Lab. Invest, biol. Univ. Madr.: Trabajos del Laboratorio de investi- 

gaciones biologicas de la Universidad de Madrid. 
Trans. Amer. Fish. Soc. : Transactions of the American Fisheries Society. New 

York. 
Trans. Amer. micr. Soc. : Transactions of the American Microscopical Society. 

Menasha, Wis. 
Trans. Amer. phil. Soc: Transactions of the American Philosophical Society. 

Philadelphia. 
Trans. Faraday Soc: Transactions of the Faraday Society. London. 
Trans. N. Y. Acad. Sci.: Transactions of the New York Academy of Science. 

New York. 
Trans, roy. Soc. Edinb.: Transactions of the Royal Society of Edinburgh. 

Edinburgh. 
Trans. R. Soc. trop. Med. Hyg. : Transactions of the Royal Society of Tropical 

Medicine and Hygiene. London. 
Trav. Inst. biol. Peterhof. : Travaux de I'lnstitut des Sciences naturelles de 

Peterhof (Biologische Institut zu Peterhof). Peterhof [Petergof]. 
Trav. Lab. zool. Sebastopol.: Travaux lu Laboratoire zoologique et Station 

biologique de Sebastopol. St-Petersbourg. 
Trav. Soc. Nat. St.-Petersb. (Leningr.) : Travaux de la Societe Imp. des 

naturalistes de St-Petersbourg (Leningrad). 
Trav. Sta. limnol. Lac Bajkal.: Travaux de la Station Limnologique du Lac 

Bajkal. 
Trop. Dis. Bull.: Tropical Diseases Bulletin. London. 
Univ. Cal. Publ. Bot.: University of California Publications in Botany. 

Berkeley. 
Univ. Cal. Publ. Zool.: University of California Publications in Zoology. 

Berkeley. 



xl LIST OF ABBREVIATIONS 

Univ. Mo. Stud.: University of Missouri Studies. Columbia. 

Unters. Bot. Inst. Tubingen.: Untersuchungen, Botanische Institut. Tubingen. 

U. S. Geol. Surv. Terr.: U. S. Geological and Geographical Survey of the 

Territories. Washington. 
U. S. Public Health Reports, see Publ. Hlth. Rep., Wash. 
Verh. dtsch. zool. Ges.: Verhandlungen der Deutschen zoologischen Gesell- 

schaft. Leipzig. 
Verh. Ges. deutscher Naturf. Artzte: Verhandlung Gesellschaft deutscher 

Naturforscher und Artzte. Berlin. (Amtlicher Bericht iiber die zweiund- 

zwanzigste Versammlung deutscher Naturforscher und Artzte in Bremen. 

Bremen.) 
Verh. naturh.-med. Ver. Heidelberg.: Verhandlungen des Naturhistorisch- 

medizinischen Vereins zu Heidelberg. 
Verh. naturh. Ver. preuss. Rheinl.: Verhandlungen des Naturhistorischen 

Vereins der Preussischen Rheinlande, Westfalens u. des Reg.-Bez. Osna- 

briick. Bonn. 
Wiss. Arch. Landw. : Wissenschaftliches archiv fiir landwirtschaft. Berlin; 

Leipzig. 
Yearb. Carneg. Instn.: Yearbook of the Carnegie Institution of Washington. 
Z. allg. Physiol.: Zeitschrift fiir allgemeine Physiologie. Jena. 
Z. Biol. : Zeitschrift fiir Biologie. Miinchen and Berlin. 
Z. ges. Anat. 1. Z. Anat. EntwGesch. 2. Z. KonstLehre. 3. Ergebn. Anat. 

EntwGesch. ; Zeitschrift fiir die gesamte Anatomie. Abt. 1. Zeitschrift 

fur Anatomie u. Entwicklungsgeschichte. Abt. 2. Zeitschrift fiir Konsti- 

tutionslehre. Abt. 3. Ergebnisse der Anatomie u. Entwicklungsgeschichte. 

Berlin. 
Z. ges. Neurol. Psychiat. : Zeitschrift fiir die gesamte Neurologie und Psychia- 

trie. Berlin. 
Z. Hyg. Infektkr. : Zeitschrift fiir Hygiene. Leipzig, 1886-91; became (Z. 

Hyg. InfektKr.) Zeitschrift fiir Hygiene und Infektionskrankheiten. Leip- 
zig. 
Z. ImmunForsch. : Zeitschrift fiir Immunitatsforschung und experimentelle 

Therapie. Jena. 
Z. indukt. Abstamm.- u. VererbLehre. : Zeitschrift fiir induktive Abstam- 

mungs- u. Vererbungslehre. Berlin. 
Z. InfektKr. Haustiere. : Zeitschrift fiir Infektionskrankheiten, parasitare 

Krankheiten u. Hygiene der Haustiere. Berlin. 
Z. mikr.-anat. Forsch. : Zeitschrift fiir mikrospopische-anatomische Forschung. 

Leipzig. 
Z. Morph. Okol. Tiere. : Zeitschrift fiir Morphologie und Okologie der Tiere. 

(Zeitschrift fiir wissenschaftliche Biologie. Berlin. Abt. A.) 
Z. Naturw. : Zeitschrift fiir Naturwissenschaften. Leipzig. 
Z. Parasitenk.: Zeitschrift fiir Parasitenkunde. (Zeitschrift fiir wissenschaft- 
liche Biologie, Berlin. Abt. F.) 



LIST OF ABBREVIATIONS xli 

Z. phys. Chem.: Zeitschrift fiir physikalische Chemie, Stochiometrie und 

Verwandtschaftslehre. Leip2ig. 
Z. Tierz. ZuchtBiol.: Zeitschrift fiir Tierziichtung und Ziichtungsbiologie. 

Berlin. 
Z. vergl. Physiol.: Zeitschrift fiir vergleichende Physiologic. (Zeitschrift fiir 

wissenschaftliche Biologic. Berlin. Abt. C.) Berlin. 
Z. wiss. Bot. : Zeitschrift fiir wissenschaftliche Botanik. Zurich. 
Z. wiss. Mikr. : Zeitschrift fiir wissenschaftliche Mikroskopie und fiir mikros- 

kopische Technik. Leipzig. 
Z. wiss. Zool.: Zeitschrift fiir wissenschaftliche Zoologie. Leipzig. 
Z. Zellforsch.: Zeitschrift fur Zcllforschung und Mikroskopische Anatomic. 

Berlin. (Zeitschrift fiir wissenschaftliche Biologic. Berlin. Abt. B [2}.) 
Z. Zcll.- u. Gewcbelehre. : Zeitschrift fiir Zellen- u. Gcwebelehre. Berlin. 

(Abt. B [1} Zeitschrift fiir wissenschaftliche Biologic, Berlin.) 
Zbl. Bakt. : Zentralblatt fiir Bakteriologie, Parasitenkunde u. Infectionskrank- 

hcitcn. Jena. 
Zool. Anz.: Zoologischcr Anzcigcr. Leipzig. 
Zool. Anz. Suppl.: Zoologischcr Anzcigcr Supplement (Bibliographia zoolo- 

gica of the Concilium Bibliographicum) . Leipzig. 
Zool. Jb.: Zoologische Jahrbiichcr. 1 Abt. fiir Anatomic. 2 Abt. fiir Sys- 

tematik. 3 Abt. fiir allgemeine Zoologie und Physiologic der Ticre. Jena. 
Zoologica. : Zoologica. Scientific Contributions of the New York Zoological 

Society. New York. 
Zool. Zbl.: Zoologisches Zentralblatt. Leipzig. 



PROTOZOA 

IN BIOLOGICAL 

RESEARCH 



CHAPTER I 

GENERAL CONSIDERATIONS 

Gary N. Calkins 

Although Protozoa are known in all parts of the world, and evidence 
is at hand that they — in some cases even the same genera — have lived and 
thrived in all periods of the earth's history, little has been accomplished 
to show how this remarkable phenomenon of longevity has been brought 
about. Within the last century, however, the matter has been the subject 
of many studies, both theoretical and experimental, although the latter, 
it must be confessed, are nearly always combined with the former. 

Life and Vitality 

For many years it has seemed to the present writer that life and 
vitality are concepts which have so often been confused that at the pres- 
ent time they are held by many biologists, and by most philosophers, to 
be synonymous. There is reason, however, especially in connection with 
the protoplasm of Protozoa, for distinguishing between them, as I have 
maintained in my recent textbook. The Biology of the Protozoa. 

It is generally recognized that life cannot be measured nor analyzed 
as such, except through its manifestations of vitality. It has long been 
known that each type of living thing has a specific organization which 
is carried on, subject to adaptations through reactions to the environment, 
or by inheritance, from generation to generation. At the present time we 
do not know what this finer organization is, but it is assumed that specific 
proteins form the basis of species differences, and that these, in combina- 
tion with water, carbohydrates, fats, and salts of different kinds, pro- 
vide the materials for metabolic activities. Thus we have the possibility 
of arriving at at least two concepts; first, the concept of the physical 
and chemical make-up, or, in general, the organization; and second, the 
concept of that same organization in action. I would apply this second 
concept to protoplasm during its activity and would limit the term 
vitality to this activity. It follows that life may be defined as specific 



4 GENERAL CONSIDERATIONS 

organization having the possibihty, or the potential of vitality. Such 
activity of protoplasm involves the interaction of its component parts 
with one another and with the environment. Life thus may be conceived 
as static, or analogous to an automobile in its garage, and dynamic, as an 
automobile in motion. A dried rotifer is almost, if not entirely static, 
but retains its organization; place it in water and it becomes dynamic 
within a few minutes. Similarly with an encysted protozoon, which, 
within its cyst walls is not freely exposed to water and oxygen but 
retains its specific organization and is apparently static and, like a dried 
rotifer, it may remain in this desiccated condition for years without losing 
its potential of vitality so that, when again placed in water, or culture 
medium, it soon emerges from its cyst, develops motile organs and other 
adult structures, and begins again its metabolic activities. The protoplasm 
within the cyst is undifferentiated, but soon after active metabolism 
begins, it becomes differentiated with structures peculiar to the species. 
After this the organization changes with each act of a metabolic nature. 

Fundamental and Derived Organization 

We have reason, therefore, to speak of a fundamental organization of 
protoplasm, characterized by undifferentiated protoplasm of a cyst, or 
of an Qgg, and of a derived organization which comes from the funda- 
mental through metabolic activity and is characteristic of the adult and 
all of its parts. It follows that death is not of necessity the absence of 
vitality but is due to the derangement or breakdown of the organiza- 
tion. 

Thus all species of Protozoa have within themselves the potential 
of an endless existence, subject, of course, to the vicissitudes of the 
daily life of the adults, each of which is the custodian of a limited portion 
of the fundamental organization. 

Some Ecological Considerations 

As well known. Protozoa may be found wherever there is moisture 
without deleterious substances and the same species may be found in the 
littoral waters of the sea and in inland fresh-water lakes, ponds, and 
pools. Although many species are cosmopolitan, they tend to accumulate 
in certain places where the environments best suit their needs; hence it 
is possible to outline certain ecological limitations, although these must 



GENERAL CONSIDERATIONS 5 

not be too closely limited. We speak, for example, of the water-dwelling 
forms, having reference to the great multitude of types which may be 
found in exposed waters of the earth's surface; or of an even greater 
number of species which live in the sea at all depths, from the surface 
down to 3,000 feet, while some Radiolaria are present in the most ex- 
treme depths of the ocean. Where salt and fresh waters mix we find 
special groups of brackish water fauna which are represented by thou- 
sands of species. 

The fresh-water species are so numerous and so varied that some help 
is gained by grouping them in "habitat groups" which are adapted more 
or less to similar environmental conditions. An attempt to classify such 
fresh-water species on an ecological basis was made by Kolkwitz ( 1909) , 
although this classification has never been widely used in protozoan 
taxonomy. It was based, in the main, on the amounts and conditions of 
organic matter and oxygen present in the water. The habitat groups 
were described by him under the terms katharobic, oligosaprobic, meso- 
saprobic, and polysaprobic types. The first are relatively rare types, being 
found in fresh-water springs, running rivers and streams, and wells 
which have little organic matter but are rich in oxygen. 

Oligosaprobic types are characterized by a small amount of organic 
matter but are rich in minerals. Hence lakes and reservoirs become the 
haunts of chlorophyll-bearing forms in particular, which often accumu- 
late to incredible numbers and frequently cause disagreeable odors and 
tastes in drinking waters and render them unpotable. 

Mesosaprobic types are the most common of all fresh-water Protozoa, 
for here active oxidation is going on and organic matter, in the presence 
of sunlight, is in all stages of decomposition. Here the microscopist finds 
his richest collecting place. 

Polysaprobic types, finally, live in waters with little or no free oxygen, 
but with an abundance of sulphureted hydrogen, carbon dioxide, and 
other products of putrefaction which are advertised by their foul odors, 
due to the gases which are formed. Here belong Lauterborn's (1901) 
"sapropelic fauna," which live, for the most part, as anaerobes, and 
which are often characterized by fantastic shapes and inability to live 
under aerobic conditions. 

Another group have an almost terrestrial habitat and may be found in 
damp moss, sphagnum, or similar environments. A few types of ciliates 



6 GENERAL CONSIDERATIONS 

and flagellates, and some testate rhizopods, may be found here, but one 
must be an optimistic collector, and an opportunist, to get good results. 

It is somewhat the same with soil-dwelling Protozoa, among which it 
is to be expected that water- dwelling forms would occasionally be found 
and interpreted as casual soil-dwelling types. Sandon (1927), however, 
has shown that there is a characteristically well-defined group of forms 
living in this environment, although, as would be expected, there is a 
wide difference in soils, both as to depth of occurrence of Protozoa and 
chemical make-up. In arable soils, Sandon finds that not only are they 
most abundant, but that they live at a depth of four to five inches and, 
for the most part, are bacteria eaters. He, with other observers, has 
described some seventy-five species of flagellates as fairly common; and 
about eighty-five species of rhizopods and ciliates which are less com- 
mon. There is not much evidence that life in the soil leads to any particu- 
lar type of morphological adaptations, but there is a possibility that species 
adapted to life deep in the soil are already partly anaerobic, and such 
forms may more easily become parasitic. 

Lackey (1925) enumerates no less than nineteen common forms of 
Protozoa which live in the sewage of Imhof tanks, while five common 
species of rhizopods, four of ciliates, and about twenty less common 
species of flagellates are occasionally found. 

So-called coprozoic forms are rarely segregated, but may be found 
more or less sporadically almost anywhere on the earth's surface. These 
are Protozoa which are taken into the digestive tract by all animals, and, 
as encysted forms, become stored up in the intestine until they are passed 
out with the feces. In water, such cysts accompanied by nutrient material 
from the feces, develop into adult and active forms which may be mis- 
taken by the unwary as entozoic parasites. So great and complete is the 
specificity of internal parasites, however, that such cysts, while not repre- 
senting parasites of the host providing the feces, may nevertheless be 
cysts of active parasites of other hosts which now pass with disastrous 
results, into the digestive tract of a new definitive host by which they 
have been eaten with contaminated food and water. 

In numbers of species there is little doubt that the free-living and 
water-dwelling Protozoa stand first, but the parasitic forms make a 
close second, for no type of animal is free from the possibility of in- 
fection by one or more species of parasitic Protozoa. 



GENERAL CONSIDERATIONS 7 

While parasitism will be dealt with by others in this volume (see 
Becker, infra, Chapter XVII; and Kirby, infra, Chapters XIX, XX), 
I will speak here merely of one or two types of adaptations which have 
arisen as a result of this mode of life. Ectoparasites and endoparasites 
have developed somewhat differently, as a result of their different modes 
of life and different needs. In common, they all possess the first great 
need of all parasites, viz. reproductive power, thus obeying a first law 
of nature to the effect that the number of offspring produced should vary 
according to the difficulties encountered in their youth and during de- 
velopment to maturity. 

Adaptations of ectoparasitic types are mainly morphological, and here 
some striking structures may be developed, as a few illustrations will 
show. All may have a special feeding advantage by being transported 
from place to place; or when attached to gills or other structures of their 
hosts, they are continuously bathed by food-bearing currents of water. At- 
tached forms live on algae, exoskeletons and appendages of arthropods, 
shells of molluscs, or gill filaments and gill bars of all kinds of water- 
dwelling animals. Thus we find species of Zoothamnium or of Lagen- 
ophrys on the legs of Gammarus, while species of Spirochona or Den- 
drocometes are on the gill filaments of the same hosts, or on Asellus. 
The suctorian Trichophrya adheres like a saddle to the gill bars of Salpa, 
while the vorticellid Ellobiophrya encircles a gill filament of the lamelli- 
branch Donax vittatus by the union of two branch outgrowths of a more 
common adhesion disc (see Kirby, Fig. XXX). The latter is almost 
always provided with hooks or suckers, or both, to form the "scopula," 
as in species of Trichodina or Cyclochaeta, parasites on Hydra. A special 
thigmotactic reaction appears to keep the ciliate Kerona pediculus on the 
surface of Hydra fusca. Such forms seem to have no ill effects on their 
hosts and scarcely qualify as parasites. Schroder calls them "epibionts." 
Real ectoparasites are rare, as a matter of fact, and they are little differ- 
ent from free water-dwelling forms in structure. They do occur, how- 
ever, especially on fish hosts. The flagellate Costia necathrix increases to 
such numbers that normal functions are impeded, and young fish are 
frequently killed. Ichthyophthirius midtifiliis bores into fish skin and 
brings about distributed ulcerations which may be fatal. 

Endoparasites may be more destructive, and, while they are relatively 
simple morphologically, they may be highly differentiated physiologically. 



8 GENERAL CONSIDERATIONS 

Knowledge of the life histories of endoparasites, particularly those of 
man, has grown amazingly, and prophylaxis has grown with it (see 
Becker, infra, Chapter XVII; and Kirby, infra, Chapter XIX-XX; and 
for immunity, see Taliaferro, infra. Chapter XVIII). 

Some Historical Facts 

Each of the myriads of species represented in this diversity of habitats 
has its own specific fundamental organization which carries the possibility 
of indefinite life in the future. We cannot visualize the conditions under 
which life came into being in times past, but we can observe, study, 
describe, and in part measure the manifestations — vitality — which have 
kept it going and enabled it to withstand all of the vicissitudes of nature, 
through upheavals, floods, droughts, and other symptoms of the might 
of nature which have played so prominent a part in the history of the 
earth's activity since the dawn of life. 

We have as a basis for such study the vast mass of knowledge which 
has accumulated since Protozoa were introduced to science by the Dutch 
naturalist Leeuwenhoek in the latter part of the seventeenth century. 
Many different kinds were soon recognized, and this recognition led to 
classifications and logical groupings in general which facilitated the dis- 
covery and descriptions of new species, modes of life, adaptations to 
changing conditions, and the like. 

Almost from the time of their discovery, the Protozoa have played 
an important part in problems of general biological interest. Spontaneous 
generation, for example, or origin of living things from nonliving mat- 
ter, which was popularly and generally believed up to the sixteenth 
century, had received hard knocks from the experiments of Redi, Spal- 
lanzani, Harvey, and other scientific men. All life from life, all living 
things from eggs [omne vivum ex ovo) supplanting the common belief 
regarding generation. 

The use of the microscope, revealing a novel and marvelous world 
of living things, gave a new lease of life to the theory of spontaneous 
generation. Appearing in containers of pure water, it was asked "How 
could such water become animated with living things if these had not 
arisen there by spontaneous generation.''" The problem, thus reopened 
with the discovery of Leeuwenhoek's animalcula, was not solved until 
near the end of the nineteenth century by the careful work of Pasteur, 



GENERAL CONSIDERATIONS 9 

Lister, and a growing school of biologists who pushed back farther and 
farther the organisms supposed to arise de novo. Lower invertebrates, 
algae and Protozoa, and finally bacteria, one after the other forming the 
fighting lines of the army of ignorance, now fell back, little by little, 
before the slow but sure advance of science. 

In connection with this theory of spontaneous generation, a novel 
conception sprang up with the observations of the French naturalist 
Buffon in 1749. This conception, expanded by Needham (1750), was 
a suggestive forerunner of the cell theory outlined by Schleiden and 
Schwann in 1839-40. The wealth of difi^erent forms of life and the 
numbers of each type in natural waters in which decomposition was under 
way, was interpreted by Buffon and Needham as the result of disintegra- 
tion of animals and plants in water and the resulting liberation of myraids 
of "organic particles" (BuflFon) of which such animals and plants were 
composed. 

Protozoology, like cytology, owes its birth and development to the 
use of the microscope. There has been much discussion and much diver- 
sity of opinion over the discovery of the microscope, meaning the com- 
pound instrument, the significant principles of which have been adapted 
and improved until the beautiful microscopes of today are the outcome. 
Woodruff (1939) writes: 

Galileo (1610) was the first to use the instrument, — but the first figures 
ever made with the aid of a compound microscope to appear in a printed 
book, were by Francesco Stelluti in 1625. But an Englishman, Robert Hooke, 
was the first to realize to the full the importance of using instruments which 
increase the powers of the senses in general and of vision in particular, and 
to express it convincingly in 1665, in a remarkable book: the "Micrographica." 
Here he described and emphasized for the first time, the "little boxes or 
cells" of organic structure, and indelibly inscribed the word "cell" in bio- 
logical literature. (Woodruff, loc. c'll., p. 2.) 

As stated above, the real discoverer of the Protozoa was a Dutch mi- 
croscopist, Anton von Leeuwenhoek (1632-1723) who, using crude 
lenses of his own make, was one of the first to apply the microscope to 
scientific investigation. His contributions to microscopic anatomy and to 
physiology, inaugurating, as they did, the invaluable services of the 
microscope in biological research, marked an epoch in the history of 
science. In a letter to the Royal Society in 1675, Leeuwenhoek wrote that 
he had discovered 



10 GENERAL CONSIDERATIONS 

living creatures in rain water whicli had stood but four days in a new 
earthen pot, glased blew within. This invited me [he continues] to view 
this water with great attention, especially those little animals appearing to 
me ten thousand times less than those represented by Mons. Schwammerdam 
and by him called Water fleas or W^ater-llce, which may be perceived in the 
water with the naked-eye. The first sort by me discover'd in the said water, 
I divers times observed to consist of 5, 6, 7, or 8 clear globuls, without 
being able to discern any film that held them together, or contained them. 
When these animalada or living atoms did move, they put forth two little 
horns, continually moving themselves. The place between these horns was 
flat, though the rest of the body was roundish, sharp'ning a little towards 
the end, where they had a tayl, near four times the length of the whole 
body, of the thickness (by my microscope) of a spiders-web; at the end of 
which a globul, of the bigness of one of those which made up the body; 
which tayl I could not perceive, even in very clear water, to be mov'd by 
them. These little creatures, if they chanced to light upon the least filament 
or string, or other such particle, of which there are many in water, especially 
if it hath stood some days, they stook entangled therein, extending their 
body in a long round, and striving to dis-intangle their tayls whereby it 
came to pass, that their whole body lept back towards the globul of the 
tayl, which then rolled together Serpent-like, or after the manner of copper 
or iron-wire that having been wound about a stick, and unwound again, 
retains those windings or turnings. This motion of extension and contraction 
continued awhile; and I have seen hundreds of these poor little creatures, 
within the space of a grain of gross sand, lye cluster'd in a few filaments. 
(From Calkins, 1901, p. 5.) 

This is the first description of a protozoon; and though the descrip- 
tion is incomplete, it undoubtedly refers to a species of Vortkella. Leeu- 
wenhoek observed several other forms, but their identity is uncertain. 

Leeuwenhoek allowed his imagination to see what his eyes could not. 

When we see [said he} the spermatic animalcula [spermatozoa} moving by 
vibrations of their tayls, we naturally conclude that these tayls are provided 
with tendons, muscles, and articulations, no less than the tayls of a dormouse 
or rat, and no one will doubt that these other animalcula which swim in 
stagnant water [Protozoa} and which are no longer than the tayls of the 
spermatic animalcula, are provided with organs similar to those of the 
highest animals. How marvellous must be the visceral apparatus shut up 
in such animalcula! (Quoted from Dujardin, 1841, pp. 21-22.) 

In a letter a year later Leeuwenhoek further says: "The fourth sort 
of little animals . . . were incredibly small; nay, so small, in my sight 
that I judged that even if one hundred of these very wee animals lay 



GENERAL CONSIDERATIONS 11 

stretched out one against another, they could not reach to the length of 
a grain of coarse sand." Later he discovered parasite Protozoa in man 
and beast, and bacteria in the human mouth. Of course Leeuwenhoek 
made many other discoveries during his long life — his studies were not 
confined to animalcules — but it is enough that he is justly regarded as 
the Father of Protozoology and Bacteriology (Woodruff, loc. cit., p. 3). 

Obviously an immense field awaited intensive study, and this was be- 
gun in a desultory way by many amateur and professional biologists dur- 
ing the closing decades of the eighteenth century, the outstanding con- 
tributions being made by O. F. Miiller in 1773 and 1786. And then 
over half a century passed before Ehrenberg, in 1838, and Dujardin, in 
1841, afforded a sufficiently broad view of the "simple" animals to justify 
the establishment of the phylum Protozoa by von Siebold in 1845 
(Woodruff, loc. cit., p. 4). 

Miiller, adopting the Linnasan nomenclature, described and named 
some 378 species, of which about 150 are retained today as Protozoa. 
His classification was the first successful attempt to bring order out of 
the heterogeneous collection of forms included under the name animal- 
cula. He used Ledermiiller's (1760-63) term Infusoria for the name of 
the entire group, which he placed as a class of the worms (see Biitschli, 
1883, p. 1129). While he eliminated many inaccuracies, he confirmed 
the substantial observations of the earlier observers, extending many of 
them to all groups of the Protozoa. He ascertained the presence of an 
anus, showed that many Infusoria are carnivorous, and observed the 
process of conjugation, his description of the latter being more accurate 
than that of any of his followers until the time of Balbiani, in 1858-59- 

Like his predecessors, Miiller included among the Protozoa many other 
organisms, placing here diatoms, nematode worms, Distomum larvae, 
and larval forms of coelenterates and molluscs. The majority of these 
miscellaneous forms were, however, properly classified before 1840, 
while finally spermatozoa (discovered by Ludwig Hamm, who is said 
to have been a pupil of Leeuwenhoek) and which had been universally 
regarded as animalcula inhabiting the seminal fluid, were withdrawn 
during the last century. 

Following John Hill (1752), MuUer did not regard the Protozoa as 
complicated animals, but considered them as the simplest of all living 
beings, composed of a homogeneous gelatinous substance, a view m 



12 GENERAL CONSIDERATIONS 

which he was followed by a majority of the "nature-philosophers," most 
of whom gave little or no attention to the Protozoa, but, accepting Miil- 
lers' work as final, based many of their speculations upon it. 

It is rather remarkable that fifty years or so later, when the biological 
atmosphere was saturated with the idea of the cell theory, the justly 
famous microscopist of Europe, C. G. Ehrenberg (1795-1876), using 
much finer achromatic lenses, should have returned to the crude view 
of Leeuwenhoek, assigning to the Protozoa a system of minute but com- 
plete organs. His conclusions on Protozoa were brought together in one 
great work, the title of which alone shows his point of view [Die 
Infusionsthierchen ah vollkoinmene Organhmen, 1838). From the sup- 
posed possession of many stomachs he gave to one of his groups the 
name Polygastrica or Magenthiere, making it a sharply defined class 
of the animal kingdom. One of the sub-classes in which these supposed 
stomachs were apparent he called the Enterodela, while all other forms he 
included as Anentera. The red pigment spots of many forms were inter- 
preted as true eyes, but as eyes could not be conceived without an ac- 
companying nervous system, he sought for nerve ganglia in different 
organisms and found what he was looking for in a species of Astasia. 
He described the "eye" in this form as seated upon a "spherical granular 
mass" which he considered as equivalent to the suprapharyngeal ganglion 
of the rotifers. The myonemes in the stalks of Vorticella, in Stentor, and 
in many other ciliates he interpreted as muscles. Pigment spheres and 
protoplasmic granules were described as ovaries, the nucleus as a testis, 
while the contractile vacuole was at first regarded as a respiratory organ 
(see Weatherby, infra, Chapter VII), 

A formidable opponent of Ehrenberg soon appeared in France — Felix 
Dujardin, who, influenced by long study of the Foraminifera, came to 
the conclusion in 1835 that these rhizopods, which up to that time had 
been classified with the cephalopod molluscs, were in reality the simplest 
of animal organisms, composed of a simple homogeneous substance to 
which he gave the name Sarcode. 

Dujardin is best known by his systematic treatise on the Protozoa which 
he published in 1841, and in which he laid the basis for the modern 
classification of these unicellular forms. The first suggestion that Proto- 
zoa might be single cells was made by Meyen ( 1839) , who compared the 
infusorian body with a single plant cell. But, according to Biitschli 



GENERAL CONSIDERATIONS 13 

(1883), the cell theory was first applied directly to the Protozoa by Barry 
(1843), who asserted that Monas and its allies among the flagellates 
were single cells, and that the nucleus found within them was the equiva- 
lent of the cell nucleus of higher forms. At the same time Barry expressed 
the view that cells increase only by division, and he compared the proc- 
esses of multiplication in Volvox and Chlamydotnonas with the cleavage 
of eggs (cf. Butschli, 1887, p. 1152). 

Barry's view was accepted, in part, by Owen, who thought, however, 
that the Infusoria could not be included with the flagellates as single 
cells because of their differentiation. It was von Siebold (1845) who 
finally asserted the unicellular nature of all Protozoa. 

Balbiani's researches on the life history of Protozoa at first led him 
into a curious error, a reminiscence, apparently, of Ehrenberg's and the 
older point of view. O. F. Miiller had observed and had correctly in- 
terpreted conjugation in different forms, but his successors down to Bal- 
biani regarded this as incorrect, maintaining that Miiller had seen only 
stages in simple division. 

Balbiani (1861) returned to Miiller's view, and clearly stated that, 
in addition to simple division, another and a sexual method of reproduc- 
tion occurs. His interpretation of the sexual organs of the Protozoa was 
given in 1858, when he maintained that the larger of the two kinds 
of nuclei of Infusoria, the macronucleus, is the ovary, and that the smaller 
one, the micronucleus, is the testis. He saw and pictured the striped ap- 
pearance of the micronucleus prior to its division and interpreted the 
stripes as spermatozoa. The eggs were said to be fertilized in the macro- 
nucleus, and then deposited on the outside where they developed into 
new ciliates. Stein at first (1859) opposed this assumption, but in the 
second volume of his work on the Infusoria (1867), misled by his own 
Acineta theory, in which certain Suctoria were thought to be stages in 
the life cycle of certain ciliates, he practically adopted it, maintaining 
however, that the young forms developed first in the nucleus and only 
later left the mother organism. Biitschli (1873) was apparently the first 
to point out Balbiani's error, and in his epoch-making work of 1876, 
after demonstrating the striped appearance of the nucleus in &gg cells 
(mitotic figure) during division, he concluded that the "stripings" which 
Balbiani held to be spermatozoa were no other than this striated con- 
dition of the nucleus during division. Butschli further showed that dur- 



14 GENERAL CONSIDERATIONS 

ing conjugation the macronucleus disintegrates, and that the parts which 
Balbiani had considered eggs are resorbed in the protoplasm, the whole 
nucleus being replaced by one of the subdivisions of the Nebenkern 
(micronucleus). Butschli's interpretation of the process of conjugation 
was equally happy. After observing that continued asexual division of 
certain forms resulted in decreased size and a general "lowering of the 
life energy," he concluded that the function of conjugation is to bring 
about a rejuvenescence {Verjungung) of the participants (see Turner, 
infra, Chapter XII). 

In the same year Engelmann (1876) obtained very similar results. 
Quite independently of Biitschli he proved the error of Balbiani's view, 
and came to the conclusion not far different from Biitschli's: "The con- 
jugation of the Infusoria," he said, "does not lead to reproduction 
through eggs, embryonic spheres, or any other kind of germ, but to a 
peculiar developmental process of the conjugating individuals which 
may be designated Reorganization {loc. cit., p. 628). 

The Use of Cultures 

Toward the end of the last century new methods of studying Pro- 
tozoa gradually evolved; at first structures, or morphological considera- 
tions were predominant; then the uses of these structures led to a general 
treatment of physiology, and a vast literature on the functions of differ- 
ent parts of Protozoa grew up. All of this, in the main, was founded 
upon observations and little was done in the experimental field of proto- 
zoan research. Staining technique aided, and great strides were made 
in knowledge of the microchemistry of all classes of Protozoa. Variations 
in structures and in functional activities became apparent, and the con- 
ception of the life cycle, first definitely outlined by Schaudinn (1900) 
as a series of forms and activities consecutively produced and performed, 
was recognized as characteristic of every species (see Kofoid, infra, 
Chapter XI). The larger fields of study thus inaugurated have become 
the starting point for many lines of research, and the single individual 
has long since ceased to be the most important goal in study of Protozoa. 

Any work on the life history, or portion of it, begins with observa- 
tions on food and feeding habits of the protozoon in question. Experi- 
ments must first be undertaken to find a suitable culture medium upon 



GENERAL CONSIDERATIONS 15 

which to grow the chosen form. The most universal of such media, in 
all probabihty, is the "hay infusion," in which hay in water, preferably 
after boihng, is allowed to stand for a certain time for bacteria to grow 
before the protozoon is placed in it. If this is a satisfactory medium, the 
organism will respond by dividing at a certain rate. After twenty-four 
hours the culture medium is replaced by fresh medium, made in the 
same way. In this manner the culture is inaugurated and carried on, and 
mass cultures are provided and sustained from the reserve individuals that 
are produced every day. In this manner material is produced for study 
of individual structures and for various activities characteristic of the 
different phases of the life cycle. 

In many inaugural cultures made in this way, the outcome is different. 
No culture is started, or at best a very poor one. The reasons for this 
are manifold. Usually in such cases the medium turns out to be a suit- 
able culture medium for some types of organisms, primarily bacteria, 
which are noxious to the protozoon under study. Hence a knowledge of 
bacteriological technique is valuable in determining the proper bacterial 
food to be used (see Kidder, hifra, Chapter VIII). 

A successful culture of a ciliated protozoon, for example, provides 
ample material for study of structures and functions; for encystment; 
or for the minutiae of cell division, conjugation, sex phenomena, and the 
like. The appearance of derived structures of all kinds may be followed 
in sequence, from their origin in the fundamental organization as it ap- 
pears in the recently encysted individual, to the active adult. Euplasmatic 
and alloplasmatic materials and their functional purposes in the cell may 
be determined, and the investigator proceeds with the confident expecta- 
tion that plenty of material will be on hand for future study. 

Very often the organism to be studied is carnivorous, and it is neces- 
sary to provide suitable food material, which must be cultivated for the 
purpose. Thus Actinobolina radians lives on Halter/a grandmella, 
Didinium nasutum on Paramecium, and Spathidium spathula on Col- 
pidium, and practically pure cultures of these food organisms must be 
kept on hand. For many purposes bacteria-free cultures must be pre- 
pared, and for this a knowledge of bacteriological technique is not only 
desirable but essential (see Hall, infra, Chapter IX; Kidder, infra, 
Chapter VIII). 



16 



GENERAL CONSIDERATIONS 



Factors Influencing Longevity 

The first factor in longevity is the set of functions which the isolated 
individual must perform in its daily life. It must react to stimuli, it 
must digest its food, it must excrete its waste matters (see Weatherby, 
infra, Chapter VII), and it must experience the processes involving 
katabolism. The euplasmatic structures having to do with these func- 
tions gradually lose their vitality, and, perhaps as a consequence of this, 
changes in activity are set up which lead to the second factor in Ion- 




Figure 1. Uronychia 
transjuga, with giant cirri, 
membranelles for swim- 
ming, ten macronuclear seg- 
ments, and single micro- 
nucleus. (After Calkins, 
1933.) 



gevity — reorganization through cell division. These changes have only 
within the last few decades been recognized and studied. 

With every activity of the euplasmatic constituents of the protoplasmic 
make-up, the derived organization is changed. These changes may be 
studied day by day, and their significance in the life history of the organ- 
ism under culture may be ascertained. Thus we learn that the macro- 
nucleus is not a portion of the fundamental organization, but is one of 
the derived organs of the ciliated Protozoa. These finer changes of the 
organization are difficult to note in the living animal, on any morpho- 
logical basis, but physiologically it is possible to show that the organiza- 
tion is not equally responsive at all stages between divisions, and the 
implication is that protoplasmic changes must have taken place. 

A merotomy experiment indicates this. A marine ciliate — Uronychia 



GENERAL CONSIDERATIONS 



17 



trans fuga (Fig. 1) — is cut transversely through the center so that ap- 
proximately half of the thirteen or fourteen beads which constitute the 




Figure 2. Uronychia trans juga, merotomy and regeneration. 1. Cell immediately after 
division, cut as indicated; 2, Fragment A of 1, three days after the operation, no re- 
generation; 3. cell cut five hours after division; 4, fragment A of 3, three days after 
operation, no regeneration; 5, cell cut at beginning of division as indicated into prospec- 
tive fragment A, B, and C; A', B', C, fragments A, B, and C twenty-four hours after 
the operation; fragment A regenerated into a normal but amicronucleate individual 
(A') ; B, C, divided in the original division plane forming a normal individual C and 
a minute but normal individual B'. (After Calkins, 1933-) 

macronucleus are left in each of the two halves, while the micronucleus 
is left undisturbed in one half. If the operation is made on an individual 
three to five hours, or less after the last previous division, the fragment 



18 GENERAL CONSIDERATIONS 

with the micronucleus regenerates perfectly, but the amicronucleate frag- 
ment, while it may live for a few days, never regenerates the missing 
structures. The same result is obtained if the organism cut is from ten 
to fifteen hours old. If, however, individuals older than this are cut in 
the same manner, an increasing percentage of complete regenerations, 
varying with increasing age, results (Fig. 2). At the time of the experi- 
ment the interdivisional period was twenty-six hours. If individuals 
were cut after twenty-one hours of age, regeneration of the amicronu- 
cleate individuals was invariable. The experiment indicates a progres- 
sive differentiation in respect, at least to the power to regenerate and, 
to that extent, a change in organization. Furthermore, if one individual 
is similarly cut while the two daughter cells at division are still con- 
nected, or shortly afterwards, there is no regeneration of the amicronu- 
cleate fragment. This indicates that the condition which underlies the 
power to regenerate is lost with processes of division and is not regained 
until the young cell has undergone a considerable period of normal 
metabolism (Calkins, 1911). This experiment was confirmed by Young 
(1922) (see also Summers, infra, Chapter XVI). 

Changes with Metabolism 

There is an accumulating amount of morphological evidence that a 
derived structure, such as the macronucleus, is constantly changing with 
continued metabolism. In Uroleptus halseyi reorganization after con- 
jugation requires from four to five days for completion. At first there 
is no chromatin in the young macronucleus, which stands out clearly 
in the young organism, and attempts to stain it after fixation are futile. 
By use of the Feulgen nucleal test, however, the chromatin reaction be- 
comes increasingly intense and the chromatin granules more distinct 
toward the end of the reorganization period; the nucleus disappears 
from sight in the living organism, but now stains intensely with any 
nuclear dye. The young macronucleus divides three times with the first 
division of the ex-con jugant and each of the daughter cells receives 
eight macronuclei, the chromatin staining deeply in all of them. This 
chromatin is in the form of discrete granules of similar form and uni- 
form size, but during the interdivisional period there appear a few 
(three to five) larger granules, which are dissolved by the Feulgen 




Figure 3A. Uroleptus mobilis Engelm. Stages in the fusion of the macronuclei prior to 
cell division. (After Calkins, 1933.) 




& 





Figure 3B. Uroleptus mobilis Engelm. Further stages in preparation for division. (After 
Calkins 1933.) 



GENERAL CONSIDERATIONS 



21 



Figure 4. Urolep- 
tus halseyi Calk. 
X-bodies, chromatin 
elimination, and nu- 
clear cleft, in prep- 
aration for division 
of the macronude- 
us. (After Calkins, 
1930.) 



method, leaving vacuoles in the nuclear substance. 
Toward the beginning of the following period of 
division, these X-granules, as they were called, be- 
come larger and more numerous, and collect, form- 
ing a disc which extends across the nucleus nearer 
one end than the other. This disc stains with acid 
dyes and is entirely dissolved by the Feulgen hy- 
drolysis. It forms the familiar Kernspalt of the 
ciliate macronucleus and its substance appears to 
act as a catalyst, for the smaller portion of the 
nucleus between the disc and the nearer end of 
the nucleus separates from the larger moiety (Figs. 
3A, 3B; Fig. 4), and eight of these large frag- 
ments now unite in the cytoplasm to form a single 
division nucleus, while the eight smaller fragments, 
with their contained X-bodies and chromatin, are 
resorbed in the cell. 

Here, then, is evidence of metabolic activity and 
change leading to the phenomena of cell divi- 
sion. 

Reorganization of the Macronucleus and 
Other Derived Structures in Ciliata 

Analogous processes of chromatin elimination, 
which numerous observers have referred to as evi- 
dence of "nuclear purification," occur in different 
ways in other ciliates. Kidder (1933) described a 
core of modified chromatin accumulating in the 
center of the macronucleus of Conchophthirus (Kid- 
deria) mytili ( Fig. 5 ) . This core condenses into a 
small deeply staining ball which, upon division of 
macronucleus, remains for a time in the connecting 
strand of the daughter nuclei, but ultimately dis- 
appears in the cytoplasm. A similar protrusion, 
referred to only incidentally by Rossolimo and 



22 



GENERAL CONSIDERATIONS 



Jakimowitch (1929), occurs in C. steenstvupii, where it is in the form 
of a finely granular substance which comes from the macronucleus and 
remains for a time between the nuclear halves after division, but ulti- 
mately disappears in the cytoplasm. 

There is reason to believe that this phenomenon has something 







Figure 5. Conchophthirus (K'ldderia) mytili. Extrusion of chromatin during division. 
(From a preparation made by Dr. G. W. Kidder.) 



to do with restoring full metabolic powers of the macronuclear chroma- 
tin, possibly by the elimination of waste products of chromatin activity. 
Another process of macronuclear reorganization during division which 
does not involve the visible elimination of nuclear substance, occurs in 
species of the families Aspidiscidae and Euplotidae of the Hypotrichida. 
At the approach of division in Aspidisca (Fig. 6), according to Sum- 
mers (1935), the area of reorganization first appears as a wedge-shaped 
cleft at the approximate center of the convex side of the C-shaped 



GENERAL CONSIDERATIONS 23 

macronucleus. A small granule in the center of the cleft represents the 
first of the reorganized nuclear material. As the cleft pushes across the 
diameter of the nucleus, the central reorganized chromatin increases in 
amount. Two separate "reorganization bands" result when the wedge- 
shaped cleft reaches the opposite side of the macronucleus. These bands 
then move in opposite directions, traversing the entire macronucleus and 
disappearing at the two ends. The increasing zone of staining chromatin 
granules, is quite different in appearance and in staining capacity from 
the chromatin in parts of the nucleus which have not been traversed 






Figure 6. Aspidisca lynceus. Origin and further history of the reorganization bands. 
(After Summers, 1935). 

by the bands. After disappearance of the bands at the ends, the nucleus 
condenses to form the typical division macronucleus of Aspidisca. Here 
again, therefore, there has been a physical change in the chromatin and a 
change that is brought about through activity of substances which form 
the nuclear cleft. 

A similar process takes place in the genus Euplotes, the reorganization 
bands of which have been studied by numerous investigators. Griffin 
(1910) was the first to describe them fully as "reconstruction bands," 
but by later writers, beginning with Yocom (1918), they have been 
known as "reorganization bands." In Euplotes the bands begin one at 
each end of the macronucleus and proceed toward the middle, where 
they meet and disappear. There is no unanimity of opinion as to what 
takes place during this passage of the bands, but all agree that some re- 
organization of the chromatin occurs. Griffin [loc. cit.) expressed the 
opinion that all of the chromatin is disolved and later re-formed without 
the erstwhile impurities. Yocom [loc. cit.), on the other hand, holds that 



24 GENERAL CONSIDERATIONS 

the chromatin does not go into solution in that part of the band known 
as the "solution plane," but appears there as closely packed granules 
and then passes to the part called the "reconstruction plane," not as 
precipitated granules, but as granules which have undergone some 
physical or possibly chemical change. Turner (1930), working on Eu- 
plotes patella, cites the absence of granules in the solution plane and re- 
gards the substance of this plane "as in the state of a colloidal solution." 
The chromatin reticulum in the center of the nucleus he interprets as in 
a continuous phase, while the karyolymph is dispersed. After action in 
the reorganization bands this condition is reversed, the chromatin gran- 
ules being in the dispersed phase and the karyolymph continuous. Phe- 
nomena of an exactly similar nature have been observed in Diophrys, 
Stylonychia, and a host of other forms so that little doubt can be enter- 
tained as to the probability that this is a highly critical period in the daily 
life of a ciliated protozoan. 

At the time of division, not only in the macronucleus but throughout 
the organism there is a wave of general house cleaning. Morphological 
parts which have been active in the metabolic reactions receive attention 
and all are built up afresh. This principle was recognized and applied 
to all parts of the cell by Wallengren in 1900 and again in 1901 when 
he wrote his illuminating paper in German. 

In the Hypotrichida in particular, in which there is no covering of cilia 
but instead motile organs called cirri, of a more complex type, Wallen- 
gren was the first to show, in great detail, that the old cirri, just prior 
to cell division, are gradually resorbed, while new ones arising from 
the cortex at an adjacent spot, grow out slowly to take their places. In 
like manner cilia are also replaced, and thus it comes to pass that, inside 
and outside, the active organs of a ciliate are composed of new materials 
derived from the fundamental organization contained in every protozoon. 

It is not only the ciliates that possess this apparent fountain of eternal 
youth; other groups of Protozoa manifest similar, if not identical phe- 
nomena. 

With few exceptions, cell division in flagellates is longitudinal, be- 
ginning as a rule at the anterior or flagellar end, the cleavage plane 
passing down through the middle of the body. As the process continues, 
the two daughter cells separate and usually come to lie in one plane, so 
that final division appears to be transverse. 



GENERAL CONSIDERATIONS 25 

As there are few details in the structure of a simple flagellate on 
which to focus attention, descriptions of division processes are practically 
limited to the history of the nucleus, kinetic elements, and the more 
conspicuous plastids. Here, in the main, are fairly prominent granules 
of different kinds, which divide as granules, and, save for the chromatin 
elements of the nucleus, without obvious mechanisms (see MacLennan, 
infra, Chapter III ) . 

In the simpler cases there is little evidence that can be interpreted to 
indicate reorganization at the time of division, and that little is con- 
fined to the motile organs. In the more complex forms, however, there 
is marked evidence of deep-seated changes going on within the cell. 

The earlier accounts of cell division in the simpler flagellates de- 
scribed an equal division of all parts of the body, including longitudinal 
division of the flagellum, if there were but one, or equal distribution 
if there were more. One by one such accounts have been checked by 
use of modern methods, until today there remains very little substantial 
evidence of the division of the flagellum. The basal body and the 
blepharoplast usually divide, but the flagellum either passes unchanged 
to one of the daughter cells, as in Crithidia, Trypanosoma, and others, 
or is resorbed into the cell. In some doubtful cases it may be thrown 
off. If the old flagellum is retained in uniflagellate forms, the second 
flagellum develops by outgrowth of the basal body or the blepharoplast. 
If the old flagellum is resorbed, both halves of the divided kinetic body 
give rise to flagella by outgrowths. Similarly if there are two or more 
flagella, one or more may be retained by each daughter cell, while the 
others, making up the full number, are regenerated. 

Reorganization is indicated, to some extent, by these cases in which 
the old flagellum is resorbed. It is still better indicated by a number 
of flagellates in which the cytoplasmic kinetic elements, as well as the 
flagella, are all resorbed and replaced by new combinations in each of 
the daughter cells. Thus in Spongomonas splendida, according to Hart- 
mann and Chagas (1910), the old blepharoplasts and two flagella 
are resorbed and new ones are derived from centrioles of the nuclear 
division figure. The phenomenon cannot be regarded as typical of the 
simple flagellates, for in the great majority the kinetic elements are 
self-perpetuating, even the axostyles, according to Kofoid and Swezy 
(1915), dividing in Trichomonas. This, however, requires confirmation. 











F r 



Figure 7. Lophomonas blattarum Janicki. Division of the nucleus and reorganization. 
(After Belar, 1926.) 



GENERAL CONSIDERATIONS 27 

An extreme case of reorganization in flagellates is apparent in the 
two species of Lophomonas (L. blattae and L. striata) first described by 
Janicki (1915). Here the parental calyx, basal bodies, blepharoplasts, 
and rhizoplasts all degenerate and are resorbed during division (Fig. 7). 
At the beginning of division, a cytoplasmic centriole divides with a con- 
necting fibril, which is retained throughout as a paradesmose. The 
nucleus emerges from the calyx in which it normally lies and moves 
with the spindle to the posterior end of the cell. The spindle takes 
a position at right angles to the long axis of the cell. Chromosomes, 



Figure 8. Chilodonella uncinatus. New pharyngeal basket and mouth, replacing old 
ones. (After MacDougall, 1925.) 

probably eight in number, are formed and divided, and two daughter 
nuclei result, each of which is enclosed in a new calyx, while new basal 
bodies and new blepharoplasts apparently arise from the polar centrioles 

(Fig-/)- 

This phenomenon in Lophomonas is strikingly similar to the re- 
organization processes occurring in one of the Chlamydodontidae — 
Chilodonella. Here, according to the observations of Enriques (1908), 
Nagler (1911), and MacDougall (1925), the old mouth of the cell 
and the oral basket of trichites are discarded and disappear in the cell, 



28 GENERAL CONSIDERATIONS 

while two new oral aggregates are provided for the daughter individ- 
uals (Fig. 8). 

In the majority of Sporozoa and Sarcodina simple division is not the 
usual mode of reproduction. Where it does occur, as in Amoebida, 
characteristic derived structures are absent and, if reorganization takes 
place, it is confined to the cytoplasm, where we have little evidence of 
change. A modified type of division, called "budding division," is widely 
spread in testacea. Here, water is absorbed by the old protoplasm, fol- 
lowed by lively cyclosis and the protrusion of a protoplasmic bud from 
the shell mouth. This bud grows, assumes the form of the parent 
organism, and secretes its own chitinous membrane on which foreign 
bodies (Arcellidae), or plates manufactured by the parent protoplasm 
(Euglyphidae), are cemented. Apart from withdrawal of old pseudo- 
podia, there is little evidence of reorganization. In Heliozoa, however, 
pseudopodia, with their axial fiilaments, are drawn in and new ones 
are formed by the daughter cells. 

In Radiolaria, Foraminifera, and Mycetozoa, indeed in the majority 
of the Sarcodina and in most Sporozoa binary fission is replaced by 
multiple division. The nuclei divide repeatedly, and a portion of the 
cytoplasm is finally parceled out to each of the nuclei. The minute cells 
thus formed leave the parent organism usually as swarm spores. Meta- 
bolic products, waste matters, and certain structures of the derived 
organization are left behind, and shells and skeletons alone mark the 
previous existence of living cells. There is often a small amount of 
protoplasm retained in these alloplasmatic products and it is not alto- 
gether fantastic to see in these remains what Weismann (1880-82) 
denied as occurring in Protozoa — viz., a corpse. 

It might seem that these methods of restoring protoplasm to its 
full vitality by processes of division are adequate to account for indefinite 
longevity of Protozoa. This, however, is not the case, with the possible 
exception of the animal flagellates, in which division in some form 
is the only means of reproduction known. In ciliates the ability to divide, 
if other methods of reorganization are prevented, gradually weakens; 
the interdivisional periods are gradually lengthened, and the degenera- 
tion of the derived structures finally results. The micronucleus may 
hypertrophy (Calkins, 1904) or disappear (Maupas, 1888), and the 



GENERAL CONSIDERATIONS 



29 



motile organs may be lost (Maupas, ibid.). In U role plus mohilis the 
chromatin of the macronucleus ultimately disappears and only a few 
X-granules remain. The protoplasm apparently dies from "old age" 

(Fig- 9). 

The effects of continued division in ciliates, if other means of re- 
organization are excluded, are clearly shown by the method of isolation 






Figure 9. Uroleptus mobilis Engelm. Old-age specimens showing the degeneration 
of the macronucleus (M) and loss of micronuclei. (After Calkins, 1919.) 



cultures. A single individual, preferably an ex-conjugant, is isolated 
in a suitable medium in a culture dish such as a Columbia isolation cul- 
ture dish. The next day such an individual, for example a U . mobilis, is 
represented by four or eight daughter individuals, the number depending 
upon two or three divisions of the original one isolated. In our practice 
five of these are isolated in separate dishes, and five independent 
lines are started, all representing protoplasm which had been a part 
of the protoplasm of the original individual isolated. A single individual 
is isolated daily from each of these five lines and a daily record of 



30 



GENERAL CONSIDERATIONS 



generations is kept for all lines. The total number of divisions is 
recorded for all lines and these are averaged for ten-day periods. The 
division rate is taken as a measure of vitality and the history of the 
protoplasm is shown graphically by plotting the ten-day averages on 
a graph in which the ordinates represent the number of divisions and 
the abscissas the consecutive ten-day periods. Such a graph for U. niobilis 
is shown in Figure 10, which is a composite graph of 23 different series, 







-I 














































li 




= 


= 




"-__ 


""l 


m 




































to 

5 














L 


















































1. 


"- 


n 








U 








L. 


"" 
































to 




20 










.10 




















i? 





Figure 10. Uroleptus mobilis Engelm. Graph representing the life history by ten- 
day periods. (After Calkins, 1933.) 



averaged for successive ten-day periods in isolation culture. It will be seen 
that there is a high initial vitality which gradually wanes through 360 
divisions during 300 days. In these isolation cultures the individuals 
do not encyst nor conjugate; hence division is the only means of re- 
organization. 

That division is not effective in checking waning vitality is shown 
by the fact that, in my experience, all such cultures of Uroleptus finally 
die, in some cases in from fourteen to sixteen months, in other cases 
in from three to ten months. 

The macronucleus is probably the most important of all derived 
organs of Uroleptus. With each division of the cell, as shown above, it 
undergoes a process of reorganization whereby the chromatin is restored 



GENERAL CONSIDERATIONS 31 

to a virile condition. This may be repeated upwards of 300 times, until 
reorganization, if it occurs, is ineffective and the protoplasm dies. Thus 
the macronucleus, like all other derived structures of the cell which 
come from the euplasmatic substances, apparently has a limited potential 
of activity. But the macronucleus may overcome this difficulty through 
the more deeply reaching phenomena in the life history, viz., endomixis 
and conjugation. 

Reorganization by Endomixis and by Conjugation 

In isolation cultures of any ciliate, if the extra individuals which 
remain over after the daily isolation is made on any day are put into 
a larger container with abundant food, a so-called "encystment test" 
or "conjugation test" is started. In my experiments these are begun 
regularly every ten days. Here the individuals multiply by division until 
there are thousands in the container. In the early stages of the life cycle 
of U. mobilis all such individuals die of starvation, but in a month 
or six weeks after the initial conjugation from which the series is 
started, such tests result in an increasing number of encystments. A 
type of breakdown which is not seen in the division phenomena is now 
manifested. The macronucleus is fragmented, and the fragments are 
distributed in the protoplasm where they ultimately disappear, while 
a new macronucleus is formed from a product of micronuclear division. 
Other structures of the derived organization are resorbed, waste matters 
and water are voided to the outside, and a cyst membrane is formed 
within which the organism may remain in a partially desiccated condi- 
tion for months. When it is recovered from the cyst and reestablished in 
isolation culture, it has an optimum vitality and passes through a com- 
plete life cycle exactly like that of an ex-con jugant. This phenomenon 
of endomixis, except for encystment, is the equivalent of endomixis 
in Paramecium aurelia as originally described by Woodruff and Erdmann 
(1914). Endomixis thus brings about a more far-reaching and more 
complete reorganization than does division; the new macronucleus aris- 
ing from a micronucleus, is provided with a new potential of vitality. 
For an up-to-date account of endomixis, see Woodruff, injra, Chap- 
ter XIII. 

An interesting phenomenon which I interpreted as analogous to endo- 
mixis occurs in the ciliate Glaucoma (Dallasia) jrontata (Fig. 11). At 



32 



GENERAL CONSIDERATIONS 



an early stage in its life history, a stage corresponding to the period 
of encystment in Uroleptus, this organism loses its normal form which 
is assumed under certain feeding conditions (see Kidder, infra, Chap- 
ter VIII), becomes elongate and cylindrical, and darts about in the 
medium with unusual speed ( Fig. 12). Soon its mouth and oral mem- 




Figure 11. Glaucoma 
(Dallasia) frontata Stokes. 
Vegetative individual. A, 
anus; BC buccal system; 
CV, contractile vacuole ; 
LS ladder system; LU left 
undulating membrane; M 
mouth of buccal cavity; 
MOT, region of motorium ; 
RU right undulating mem- 
brane; T, "tongue" in buc- 
cal cavity. (After Calkins 
and Bowling, 1929.) 



branes are resorbed and it divides into two cylindrical individuals. 
These divide into four, the four into eight, and the eight into sixteen, 
all without intervening growth. The last-formed individuals have no 
resemblance to the original parent form, but each has a macronucleus 



GENERAL CONSIDERATIONS 



^3 



and a micronucleus which has undergone maturation divisions and is 
haploid as to chromosomes. The final sixteen are associated in pairs, 
which become surrounded by a cyst membrane within which they fuse 
(Calkins and Bowling, 1929). For other variations in the phenomena 
of endomixis, see Diller, 1936; and especially Woodruff, infra, Chap- 
ter XIII. 

This unusual phenomenon in Dallasia, which was not encountered by 
Kidder in his studies on Glaucoma, finds its closest parallel in a group 




Figure 12. Glaucoma (Dallasia) jrontata Stokes. History of normal vegetative in- 
dividual with successive divisions resulting in copulating gametes and encysted zygotes. 
(After Calkins and Bowling, 1929.) 



of ciliates in each of which the life history is unique. This group includes 
the ectoparasite Ichthyophthirius multifilih, according to Buschkiel 
(1911) and Nerescheimer (1908), and O patina ranarum, according to 
Metcalf (1909) and Nerescheimer (1907). With these forms we have 
good evidence, together with Dogiel's "transformation of a male pro- 
nucleus into a spermatozoon" in the Ophryoscolecidae, that the intra- 
cellular micronuclei forming pronuclei are a reminiscence of a brood 
of gametes (Dogiel, 1923; see also Turner, injra. Chapter XII). 



34 



GENERAL CONSIDERATIONS 



The longevity of a ciliate's protoplasm in the past is shown by the 
fact that it is before us today and it has a possibility of indefinite 
longevity in the future. We know very little about the secrets of proto- 
plasmic organization which underlie continued life, but we can analyse 
some, at least, of the conditions under which it maintains its animation. 
A proper environment and adequate food are essential factors. These 
lead to growth and to reproduction by division. We have seen that these 
latter bring about a reorganization and a return to full metabolic ac- 




Figure 13. U role plus 
mobilis Engelm. Conjuga- 
tion and merotomy. (After 
Calkins, 1921.) 



tivity. The periodic restoration of these processes might well be enough 
to ensure protoplasmic longevity, as seems to be the case with the 
animal flagellates. At endomixis the ciliate macronucleus follows the 
fate of other parts of the derived organization; a new one is formed 
from the fundamental organization, and the result is, again, increased 
vitality of the protoplasm. This phenomenon, recurring every thirty 
days, ensured the longevity of Parameciuni aurelia in Woodrufl^'s hands 
for many years and through thousands of generations by division. As 
Woodruff states, it may be adequate, indeed, to ensure indefinite life 
in the future, as division alone is apparently adequate for animal 
flagellates. 

In a conjugation test made with Uroleptus, the results showed that 
if the series is sixty or more days old, the individuals multiply by 



GENERAL CONSIDERATIONS 35 

division until there may be thousands in the container. If conditions are 
appropriate, this massing may be followed by an epidemic of con- 
jugations (see Sonneborn, /nfra, Chapter XIV). In this process two 
individuals fuse at the anterior ends and remain united for approxi- 
mately twenty-four hours ( Fig. 13). They then separate and each of 
the ex-con jugants begins a process of reorganization which lasts for 
four or five days. If such an ex-conjugant is isolated and fed, it will 
give rise to a new series and its progeny will pass through all the stages 
of vitality that the parent series passed through. At the outset its vitality 
will be approximately the same as the original vitality of the parent 
series, but it will be greater than that of the parent series at the time 
of conjugation. It passes through the same history of waning vitality, 
and its protoplasm finally dies, although this may be months after the 
death of the parent protoplasm. 

These experiments with Uroleptus, continued for more than ten 
years and with 146 different series, always gave the same result. The 
length of life of each series varied from three months to over a year 
and all were descendants of a single bit of protoplasm making up the 
body of one Uroleptus cell. 

There is little reason to doubt that some change in the protoplasmic 
make-up occurs with continued metabolic activity through many suc- 
cessive generations by division. In isolation cultures it is manifested by 
an increasing time interval and by a change in the physical properties 
of the protoplasm whereby fusion of cells, total or partial, is possible. 
A type of breakdown, not manifested early in the series, is set up. The 
cortex is liquified, in most cases in the region of the peristome, and two 
individuals fuse in conjugation. In some cases the entire cortex is thus 
modified, and individuals will fuse at any point on the periphery. I 
have often referred to one case where no less than nine Paramecium 
caudatum were thus fused into one amorphous mass. 

The stimulus of normal fusion results in activities not manifested 
before. The micronuclei divide as they do in endomixis, and their 
division involves reduction in number of chromosomes and in the 
formation of pronuclei which meet and fuse. These copulating micro- 
nuclei, as pronuclei, are usually interpreted as a reminiscence of an 
ancestral gamete brood which is realized in Dallasia [Glaucoma) fron- 
tata, Opalina, and in gregarines. In the latter, as is well known, each indi- 



36 GENERAL CONSIDERATIONS 

vidual of a syzygy forms a brood of gametes which copulate with 
similar gametes from the other individual of the pair (see Turner, infra, 
Chapter XII; and Kofoid, infra, Chapter XI). 

Coming at the end of a vegetative period during which single or 
multiple divisions may have occurred, such gamete broods indicate a 
change in organization which, if copulation does not occur, results in 
death of the gametes. In other words gametes are so specialized that 
they require a complemental combination to ensure normal metabolism 
and life. In some gametes differentiation has been in the direction of 
greater constructive activity, resulting in food-stored macrogametes 
equivalent to egg cells; in others in the direction of greater katabolic 
activity, resulting in a brood of microgametes equivalent to spermatozoa, 
as in Coccidiomorpha. 

The happenings at conjugation need only be recapitulated, for the 
phenomena are so well known that description here is unnecessary (see 
Turner, infra, Chapter XII, for detailed accounts). In U role p/ us, shortly 
after fusion of the anterior ends, the micronuclei of both individuals 
begin a series of maturation divisions, and the third division gives rise 
to the gametic nuclei. One of a pair of these remains in situ, while the 
other, or migrating pronucleus, passes through the protoplasmic bridge, 
unites and forms a fertilization nucleus by fusing with the quiescent 
pronucleus of the other conjugant. Thus a mutual fertilization occurs, 
the two migrating pronuclei in Uroleptus passing each other at the 
apex of the united pair of cells (Fig. 13). After this is accomplished, 
the two individuals separate, the amphinucleus of each conjugant di- 
vides twice, and one of the products forms a new macronucleus, which, 
after four or five days, is ready to divide. Another product forms a 
new micronucleus. In the meantime the old macronucleus begins to 
degenerate and is ultimately resorbed in the cytoplasm. The other 
structures of the derived organization of the old organism are resorbed 
and replaced by new ones. The young organism, with this new set 
up, starts a new life cycle with an optimum of vitality (Fig. 14). 

Through these activities of conjugation, an old protoplasm with low 
vitality is made over out of its own contained substances, into a new pro- 
toplasm with high vitality. What explanation of this remarkable phenom- 
enon can be given? The only apparent difference between the happen- 
ings at division and at conjugation are: (1) fertilization, and (2) 



GENERAL CONSIDERATIONS 



37 



loss and replacement of the old macronucleus, and so forth. Renewed 
vitality may be due to one or to both of these phenomena. Conjugation 
differs from endomixis mainly in gelatinization of the cortex and in 



.«^ 








Figure 14. Uroleptus mobilis Engelm. Formation of new macronucleus after conjuga- 
tion. 1. First metagamic or zygotic division of the amphinucleus; 2. One of the progeny 
of this division dividing again; 3, 4, 5, telophase stages of this divison resulting in a 
new macronucleus (above) and a degenerating nucleus (below) ; 6 to 10, stages in the 
differentiation of the new macronucleus and disintegration and resorption of the old 
macronucleus. In 10, two new micronuclei are in mitosis preparatory to the first division 
of the ex-conjugant. (After Calkins, 1919.) 

amphimixis. Fertilization, with its sequelae of hereditary possibilities, 
is a highly important result (see Jennings, injra, Chapter XV; and 
Sonneborn, injra, Chapter XIV). It is also assumed to be the raison 



38 GENERAL CONSIDERATIONS 

d'etre of all sorts of subsequent peculiarities, but for the phenomenon 
of increased vitality under consideration, a very simple experiment with 
Uroleptus mohilh shows that fertilization with amphimixis has little to 
do with the problem. A conjugating pair is cut across the apex with a 
scalpel. One individual of the two thus separated is fixed and stained, to 
show the stage of conjugation when cut, while the other one is cultivated 
in an isolation culture dish. The experiment is particularly striking when 
the two wandering pronuclei are cut off with the apical piece ( Fig. 13). 
The cultivated individual goes through exactly the same processes as 
though conjugation had been completed in the usual manner, and a 
normal life cycle results. Amphimixis, however, had been prevented; 
conjugation apparently had been transformed into endomixis (Calkins, 
1921). Here, however, the possibility remains that one of the super- 
numerary micronuclet of the amputated individual may divide and one 
part may unite with the pronucleus already present. 

The loss and replacement of the old macronucleus, together with 
that of the old derived structures generally remains as a possible ex- 
planation. This phenomenon is common to endomixis, conjugation, and 
conjugation merotomy, and in all cases a renewed vitality results. This 
does not happen with division, although, as shown above, characteristic 
changes occur in the macronucleus. With its disintegration and re- 
sorption in the cytoplasm, a new supply of nucleoproteins and other 
chemical compounds useful in metabolic activities are distributed in the 
cell, and these may be potent factors in the new vitality. Added to these 
is the fact that an entirely new and powerful organ of the cell — the 
macronucleus — has been supplied from the fundamental organization. 

The majority of the derived structures of the cell have a relatively 
short life, being resorbed and renewed at division (cirri and other 
motile organs; others, such as membranelles and oral structures, appar- 
ently have a longer life. Of all derived structures the macronucleus has 
the longest life; it divides at cell division and may remain functional 
through entire life cycles, including many hundreds of divisions. It is 
probably the chief metabolic agent of the cell, yet it apparently lacks 
the power of continued life which the micronucleus possesses. It is 
essentially somatic in character, and, like the soma in metazoa, it ulti- 
mately wears out; if not replaced, the rest of the cell, including the 
micronucleus depending on it, dies with it. It is quite possible, indeed 



GENERAL CONSIDERATIONS 39 

I regard it as probable, that the gradual deterioration of this important 
organ of the ciliate cell, is the underlying cause of waning vitality and 
ultimate death of protoplasm in isolation cultures (Fig. 10). 

With parasitic forms there is probably the same underlying variation 
in vitality, but there is not the same possibility of measuring it, at least 
not in any direct way. 

Remarkable as these phenomena are, they leave us cold so far as the 
matter of protoplasmic vitality is concerned. The interpretations pre- 
sented here are, after all, essentially mechanistic, and even with the 
ciliates the phenomena described are by no means universal, while in 
some groups of Protozoa they are not shown at all, or else only in a 
vague and indefinite manner. In ciliates there is one organoid of the 
cell, the micronucleus, which transcends all other structures of the cell, 
and, although it is apparently not functional except in heredity and 
activities connected therewith, such as regeneration, and so forth, it 
does appear to be the most essential morphological element of the 
fundamental organization. Its agent in metabolism is the macronucleus, 
which is derived from it. For the secret of life and longevity in ciliates 
we must turn to this inconspicuous and often overlooked structure of 
the cell and of the cyst. 

In other groups of the Protozoa, the homologue of this important 
element of the cell lies in the usually single nucleus. Furthermore, in 
the micronucleus it is probably the chromatin content that gives it its 
power; and, in other groups than the ciliates, it is the chromatin con- 
tent that makes the nucleus what it is. The value and importance of 
chromatin is seen by the meticulous care with which it is distributed 
to daughter cells and to progeny generally, while the maturation divi- 
sions bespeak its significance in heredity. The secret of life and vitality 
must thus be sought not in the daily activities of living things, but in 
that enigmatical substance — chromatin — which is about us in all living 
things, including ourselves. That secret may never be disclosed. 

Literature Cited 
Balbiani, G. 1859. Du role des organes geneateurs dans la division spent, des 

infusoires cilies. J. Physiol. Pathigen., Ill: 71-87. 
1861. Recherches sur les phenomenes sexuelles des infusoires. J. Physiol. 

Pathigen., : 102-30, 194-220, 431-48, 465-520. 
Barry, M. 1843. On fissiparous generation. Edinb. New phil. Jour., 35: 205- 

20. 



40 GENERAL CONSIDERATIONS 

Belar, K. 1926. Der Formwechsel der Protistenkerne. Ergebn. Zool., 6: 235- 

654. 
Buffon, G. L. de. 1749. Histoire naturelle generale et particuliere. 
Buschkiel, A. L. 1911. Ichthyophthhius mult/fiU/s. Arch. Protistenk., 21: 61- 

102. 
Biitschli, O. 1873. Einiges uber Infusorien. Arch. mikr. Anat., 9: 657-78. 

1876. Studien iiber die ersten Entwicklungsvorgange der Eizelle, die 

Zelltheilung, und die Konjugation der Infusorien. Abh. senckenb. naturf. 
Ges., 10: 213-462. 

1887-89. Bronn's Klassen, und Ordnungen des Thier reichs. Erster Band. 

Protozoa. Leipzig. Pp. 1-2028. 
Calkins, G. N. 1901. The Protozoa. New York, 1901. 
1904. Studies on the life history of Protozoa. IV. Death of the A-series. 

J. exp. Zool., 1: 423-59. 

1911. Regeneration and cell division in Uronychia. J. exp. Zool., 10: 

95-116. 

1919. Uroleptus mohilh Engelm. I. History of the nuclei during division 

and conjugation. J. exp. Zool., 27: 293-357. 

1921. Uroleptus moh'ilh Engelm. Effect of cutting during conjugation. 

J. exp. Zool., 34: 449-70. 

1930. Uroleptus halseyi. II. The origin and fate of the macronuclear 

chromatin. Arch. Protistenk., 69: 151-74. 

1933. The Biology of the Protozoa. 2d ed., Philadelphia. 

1934. Factors controlling longevity in protozoan protoplasm. Biol. Bull. 

Woods Hole, 67: 410-41. 

Calkins, G. N., and R. Bowling. 1929. Studies on Dallasia frofitata Stokes. 
II. Cytology, gametogamy and conjugation. Arch. Protistenk., 66: 11-32. 

Diller, Wm. F. 1936. Nuclear reorganization processes in Paramecium aurelia, 
with descriptions of autogamy and "hemixis." J. Morph., 59: 11-51. 

Dogiel, V. 1923. The transformation of a male pronucleus into a sperma- 
tozoon. Zool. Lab. Univ. Petrograd, 1923. 

Dujardin, F. 1835. Observations sur les rhizopodes et les infusoires. C. R. 
Acad. Sci. Paris, Nov. 1835, pp. 338-40. 

1841. Histoire naturelle des zoophytes infusoires. Paris. 

Ehrenberg, C. G 1938-39. Die Infusionsthierchen als vollkommene Organis- 
men. Leipzig. 

Engelmann, Th. W. 1876. Uber Entwicklung u. Fortpflanzung der Infusorien. 
Morph. Jb., 1: 573-635. 

Enriques, P. 1908. Die Conjugation und sexuelle Differenzierung der In- 
fusorien. Arch. Protistenk., 12: 213-74. 

Griffin, L. E. 1910. Euplotes tvorcesteri, sp. nov. II. Division. Philipp. J. Sci., 
5: 291-312. 

Haas, G 1933. Beitrage zur Kenntnis der Cytologie von Ichthyophthirius 
multifil/is. Arch. Protistenk., 81: 88-137. 



GENERAL CONSIDERATIONS 41 

Hartmann M., and C. Chagas. 1910. Flagellaten Studien. Mem. Inst. Osw. 

Cruz, 2, fasc. I. 
Hill, John. 1752. History of Animals. 3d vol. of a gen. Nat. Hist., 1748-52. 
Hooke, Robt. 1665. The Micrographia. London. 

Janicki, C. 1915. Parasiten Flagellaten, Teil 2. Z. wiss. Zool., 112: 573-691. 
Kidder, G. W. 1933. Studies on Concophthhius 77iytili de Morgan. I. 

Morphology and division. Arch. Protistenk., 79: 1-24. 
Kofoid, C. A., and O. Swezy. 1915. Mitosis in Trichomonas. Proc. nat. Acad. 

Sci., Wash., 1: 315-21. 
Kolkwitz, R., and M. Marsson. 1909. Oekologie der tierischen Saprobien. 

Int. Rev. Hydrobiol., 2 : 
Lackey, J. B. 1925. Studies on the biology of sewage disposal. The fauna of 

Imhof tanks. Bull. N. J. agric, Exp. Stas., No. 417. 
Lauterborn, R. 1901. Die "Sapropelische" Lebewelt. Zool. Anz., 24: 50-55. 
Ledermiiller, M. F. 1760-63. Mikroskopische Gemiiths. u. Augenergortzungen. 

Nurnberg, 1760: 88. 
Leewenhoek, A. van. 1676. Observations concerning little animals by him 

observed in rain, well, sea, and snow water wherein pepper had lain 

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MacDougall, M. S. 1925. Cytological observations on gymnostomatous ciliates, 

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Zool. Exp. gen., (2) 7, 8. 
Metcalf, M. M. 1909 Opalina. Its anatomy, etc. Arch. Protistenk., 13: 195- 

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Meyen, J. 1849. Einige Bemerk. iiber den Verdauungsapparat der Infusorien. 

Arch. Aanat. Physiol. Lp. z., 1839: 74-79. 
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Miiller, O. F. 1773. Vermium terrestr. et fluviatil. s. animal, infusor. etc. Haf- 

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1786. Animalc. infusoria, fluviat. et marina, etc. (posth.) Hafniae et 

Lipsiae. 
Nagler, K. 1911. Caryosom u. Centriol Beimteilungsvorgang von Chilodon 

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Physiol. Miinchen, 22: 
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Rossolimo, L. I., and Frau K. Jakimowitsch. 1929. Die Kernteilung bei Con- 

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42 GENERAL CONSIDERATIONS 

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Young, Dixie. 1939. Macronuclear reorganization in Blepharisnia undulans. 

J. Morph., 64: 297-347. 



CHAPTER II 

SOME PHYSICAL PROPERTIES OF THE PROTOPLASM 
OF THE PROTOZOA 

H. W. Beams and R. L. King 

Introduction 

DujARDiN in 1835, a little over a century ago, was the first to care- 
fully describe the physical properties of protozoan protoplasm which 
he termed "sarcode," although other earlier observers had seen and 
drawn living amoebae. For instance, Rosel von Rosenhof drew an amoeba 
in 1755, O. F. Miiller described living amoeba in 1773 and Ehrenberg, 
a pioneer protozoologist, undoubtedly observed living protozoan proto- 
plasm. However, none of these observers emphasized the protoplasm as 
the living substance as did Dujardin. In addition, Dujardin's description 
of protoplasm was so accurate that his definition of it as a "living jelly, 
glutinous and transparent, insoluble in water, and capable of contract- 
ing into globular masses and of adhering to dissecting needles so that it 
can be drawn out like mucus" can be little improved upon today (see 
Faure-Fremiet, 1935). 

Perhaps no group of animals has served as the basis for so many 
and so extensive studies on the structure of protoplasm as the Protozoa. 
This is, no doubt, in part due to the fact that many of the earlier 
workers labored under the assumption that the "simplest type" of proto- 
plasm should be looked for in the "lower forms" of animals. In addi- 
tion many of the Protozoa are comparatively large and discrete cells, 
thus offering little mechanical difficulties to direct microscopic observa- 
tions upon their living structure. 

In a modern discussion of the physical properties of protoplasm, one 
must bear in mind the fact that the manifold chemical and physiological 
properties of living matter are intimately connected with the structure of 
protoplasm. That is, one must conceive of a system which is kept in 
existence, in spite of the fact that the katabolic phases of energy ex- 
change tend to destroy its integrity; this involves a continuous auto- 



44 PROTOPLASM OF PROTOZOA 

matic replacement of worn-out parts or the growth of new ones. Chemical 
processes must often be definitely localized in a small region within the 
framework of a single cell. However, visible differentiation usually does 
not take place within a single cell in the Metazoa to such an extent as 
in the Protozoa, where we have a whole series of special differentiated 
organelles for performing particular functions. 

A protozoan is usually regarded as both an organism and a cell; in 
the Metazoa protoplasmic differentiation tends to be irreversible, and 
in Protozoa to be reversible, so that the most highly differentiated cells 
ever observed are found as individual Protozoa. This extreme differentia- 
tion is, of course, surprising to one accustomed to the usual structural 
simplicity of metazoan cells, and is probably the main reason for the 
position taken by Dobell (1911) and others who deny the cellular 
nature of the Protozoa. Several degrees of permanency of differentiation 
may be distinguished among the Protozoa, although of course the groups 
are not mutually exclusive. Thus we may distinguish: (1) temporary, 
completely reversible structures or differentiations of the protoplasm, 
such as pseudopodia and spindle elements; (2) differentiations usually 
irreversible and lasting throughout the life of the organism, which may 
be the seat of active chemical energy changes, such as cilia, flagella, and 
myonemes, or, with little chemical changes, such as morphonemes and 
pellicle; and (3) differentiations formed by protoplasm which are 
chemically distinct from it, such as shells and secretions. 

Every time a protozoan divides, conjugates, or encysts there is a 
tendency toward dedifferentiation, followed by reorganization, so that 
the individual emerging from these processes often has developed a 
new set of structures derived from the fundamental structure. These 
changes must be accompanied or caused by physical realignments of 
protoplasmic elements, of which little is known. Perhaps in no other 
phylum of animals can such a wide range of type of mitosis and 
cytokinesis be found as exists in the Protozoa. The morphological de- 
tails of the division process in these forms are fairly well known, and 
evidence is continually accumulating which shows that mitotic and 
cytokinetic mechanism does not differ fundamentally from that of 
higher forms. 

In the Protozoa, as in other types of cells, little is actually known 
regarding the initial stimulus that starts the organism to divide, to 



PROTOPLASM OF PROTOZOA 45 

undergo certain sexual phenomena, or to form cysts. However, environ- 
mental conditions, such as amount and kind of food present, tempera- 
ture, and so forth, have been generally observed to affect the rate at 
which these phenomena take place. Since Protozoa under normal condi- 
tions seem to be limited to a more or less uniform maximum size for 
a given species, various suggestions have been made to the effect that 
the stimulus for division is in part controlled by definite ratio of volume 
to body surface, or to a possible ratio of nuclear volume to cytoplasmic 
volume. However, whatever may prove to be the explanation of this, 
it is an observed fact that in most cases conditions suitable for most 
rapid growth are the same as those best suited for most rapid division. 
On the other hand, relatively unfavorable conditions, such as scarcity 
of food or the concentration of metabolites, have been observed to in- 
duce sexual activity in the various Protozoa (see Giese, 1935, for litera- 
ture). Practically nothing can be said of the physical changes under- 
gone by the protoplasm of the organisms just before they start division, 
conjugation, or endomixis, except that before division they may shorten 
and thicken {Uroleptus, Calkins, 1919), or become spheroidal in form 
{Amoeba, Chalkley, 1935). Such observations possibly indicate a change 
in viscosity, the nature of which awaits investigation. 

In certain of the hypermastigote flagellates Cleveland (1934, 1935) 
has found one of the best materials for the study of the cytology of 
cell division, not only in the Protozoa but of cells generally. Here the 
mitotic spindle, with its chromatic and achromatic parts, is clearly seen 
in the living condition; it may also be easily preserved and stained by 
ordinary techniques. In addition, experiments on pulling the centrosomes 
demonstrate the elasticity and contractility of the extranuclear chromo- 
some fibers and those of the central spindle. Accordingly, in this ma- 
terial the various elements of the mitotic apparatus must be considered 
real and therefore demand careful consideration in any discussion of the 
mechanics of mitosis, not only in these forms but in astral types of 
mitotic divisions generally. Furthermore, a study of the physical prob- 
lems involved in the origin and nature of the centrosomes, spindle 
fibers, asters, degeneration and reorganization of certain of the locomotor 
organs, as well as the correlated mechanics of cytokinesis in these forms 
would be most valuable. However, for the present we can only assume 
that the physical-chemical changes giving rise to these various struc- 



46 PROTOPLASM OF PROTOZOA 

tures are similar to those which occur in the mitotic divisions of cells 
of higher forms. For a discussion see Gray, 1931; Heilbrunn, 1928; 
and Chambers, 1938. 

Properties of Protoplasm as Exhibited in Amoeba 

To obtain a concept of protoplasmic structure no better course can 
be followed than to secure a microscope, some amoebae, and study them 
under relatively high power. Efforts to obtain this information from the 
literature alone often lead to confusion, because of the wide variety of 
terminology used by the various workers on the structure of amoebae. 
Careful microscopic examination of the protoplasm of Amoeba proteus 
in locomotion will reveal that it is composed of an outer, thin, colorless, 
hyaline layer which is for the most part optically structureless, and an 
inner granular region which makes up the greater portion of the 
animal. A detailed study of the granular portion will reveal that it is 
composed of a colorless continuous phase (hyaloplasm), in which are 
suspended crystals and granules of various sizes and shapes, nucleus, 
food vacuoles, contractile vacuole, refractive bodies, oil globules, and 
possibly other inclusions. Long intervals of observation on these struc- 
tures will show that some of them seem to be permanent and self- 
perpetuating while others are only transitory. The question that naturally 
arises in this connection is, which are living and which are nonliving 
bodies? A further discussion of this point will be given elsewhere. 

The question may now be asked, how are the various microscopically 
visible inclusions of Amoeba protoplasm kept in suspension? This may 
be because their density is only slightly different from that of the sur- 
rounding protoplasm and because of the viscosity of the protoplasm. 
Mast and Doyle (1935a) have presented evidence that the refractive 
bodies, which are the heaviest inclusions of the protoplasm, are found 
near the lower surface in living amoebae in which the protoplasmic vis- 
cosity is low; that they are distributed by protoplasmic flow has been 
demonstrated by Mast and Doyle (1935b). Upon centrifuging, the 
cytoplasmic constituents of amoeba are layered out, in order of their 
relative specific gravity, with the refractive granules at the centrifugal 
pole. Upon recovery pseudopods form first at or near the lighter end, 
and the heavy refractive bodies then flow forward followed by the other 
layers of constituents until all are thoroughly mixed. 



PROTOPLASM OF PROTOZOA 47 

The granular region of an amoeba is made up of an outer stationary 
layer, which forms a hollow cylinder through which an axial stream of 
protoplasm flows. The outer stationary layer has been called the ecto- 
plasm and, more recently, because of its firm consistency, plasmagel; 
the axial flowing layer, endoplasm, and, more recently, because of its 
fluid consistency, plasmasol. More difficult to observe, because of its 
extreme thinness, is the surface layer, or plasmalemma, just external to 
the clear hyaline layer. 

Ordinary methods of fixation often result in an apparent loss of the 
constituents which may be seen in the living cell; however, by the use 
of suitable fixing methods Mast and Doyle (1935a) were able to show 
that in A. pro tens the cytoplasmic constituents may be preserved in a 
form scarcely distinguishable from the living condition, except for the 
nuclear granules and the water-soluble salts. Since protoplasm is an un- 
stable intimate association of salts in solution, proteins, fats, and other 
materials which may give it a fibrillar or an alveolar appearance, we 
cannot hope to preserve it unchanged. Indeed, we may not even ob- 
serve it in the living condition unchanged, as change is a universal 
characteristic of protoplasm. It must be constantly kept in mind that 
what is observed in a permanent fixed preparation is not living material, 
but likewise it must also be recognized that while the fixed material is 
not protoplasm, it is at least a significant artifact derived from proto- 
plasm, since it has had its origin in the coagulation of proteins and 
other significant elements of living material. Most of the established 
facts of cytology have been first observed in fixed material, and later 
corroborated by a study of the living. However, it should be pointed 
out, from the works of Hardy and of Fisher, that probably many of 
the older theories of protoplasmic structure, such as the granular, fibril- 
lar, alveolar, and reticular, have been based in part, at least, on artifacts 
induced by the methods employed. 

Probably the most significant feature of the protoplasm of Amoeba 
is its ability to change from a relatively fluid state to a more solid jelly- 
like condition. Upon mechanical agitation, such as occurs in trans- 
ferring an amoeba to a slide or by tapping on the slide, the organism 
may assume a globular form which may have short projecting pseudo- 
podia. If the temperature be raised to 30° C, the projections may dis- 
appear (K. Gruber, 1912). This means that the protoplasm present 



48 PROTOPLASM OF PROTOZOA 

has become relatively fluid, because only a liquid immiscible with the 
surrounding medium shows the globular form which is essential in the 
principle of minimal surfaces. This change in consistency may be 
brought about by mechanical agitation (Chambers, 1921) and has been 
experimentally studied by Angerer (1936), who presents data to show 
that mechanical agitation of amoebae causes a decrease in viscosity in the 
plasmagel [A. proteus) and an initial decrease followed by an in- 
crease in viscosity in the plasmasol [A. duhid) . Extreme mechanical 
agitation according to Angerer, leads to a minimum viscosity of the 
plasmagel, which is eventually followed by the disintegration of the 
organism, the substance of which mixes with the surrounding medium. 

If enough pressure is applied to the cover glass, the amoeba will 
burst, and it may be observed, providing the injury is not too extensive, 
that an effort is made on the part of the protoplasm at the region of 
rupture to form a water-insoluble membrane, thus inhibiting further 
mixing of the protoplasm with the water. This process has been re- 
cently termed "the surface precipitation reaction" by Heilbrunn (1928). 
If, however, the injury has been sufficient so that the interphase be- 
tween the ruptured protoplasm and the surrounding medium is too 
great, the surface precipitation reaction is not sufficient to prevent a 
complete dissolution of the amoeba into the water. 

Small hemispheres of clear protoplasm (the incipient pseudopodia) 
usually appear quickly upon the surface of a quiescent spheroidal 
amoeba in the form of liquid extrusions from the main mass of proto- 
plasm. Into these extrusions, which gelate equatorially, a central flow 
of granular protoplasm may be seen streaming forward from the amoeba 
in the direction of the advancing pseudopodium, which constantly moves 
distally. As the pseudopodium advances, it may be seen to consist of an 
outer cylinder of motionless protoplasm and an inner streaming fluid 
protoplasm. That the cortical layer is gelated is indicated by the fact 
that its granules and other inclusions are stationary. As the central 
stream of flowing protoplasm (endoplasm or plasmasol) reaches the 
hyaline cap, it moves peripherally in all directions and gelates (i.e., 
becomes ectoplasm or plasmagel). At the other end of the amoeba the 
plasmagel becomes plasmasol and passes forward as a fluid core through 
the cylinder of plasmagel (Mast, 1926). The cause of the forward 
flow of protoplasm is obscure, but the suggestion has been made by 



PROTOPLASM OF PROTOZOA 49 

Hyman (1917) and many others that it is due to the tension exerted 
upon the fluid plasmasol by the elastic plasmagel. 

There has been much discussion concerning the physical characteristics 
of the outermost layer, or plasmalemma, of different species of amoeba. 
In certain forms, such as A. verrucosa, the pellicle may be lifted with 
microdissection needles and stretched, apparently without injuring the 
organism in any way (Rowland, 1924c). Seifriz (1936) also found 
a thin outer membrane on A. proteus which was resistant, elastic, and 
highly viscous except at the advancing tip of the pseudopodia. Likewise 
Chambers (1924) was able to lift the plasmalemma in A. proteus by 
injecting water beneath it, thus causing large blisters to form between 
the plasmalemma and the underlying surface which burst upon punctur- 
ing, leaving the pellicle collapsed. Mast (1926) caused blisters to 
appear by local pressure; further pressure caused disruption of the 
pellicle, the frayed ends of which were clearly observed. 

Practically all investigators are agreed that the plasmalemma moves 
forward, at least on the upper surface of a moving amoeba. In addition, 
the method of formation of new food vacuoles requires a structure 
of the plasmalemma of such nature as to form new surfaces immediately 
by the replacement of large areas of the plasmalemma which have been 
used in forming the boundary of the food vacuoles (Schaefl^er, 1920). 

Immediately beneath the outer layer, or plasmalemma, is the clear 
hyaline layer. That this layer is fluid may be shown by the fact that it 
contains scattered granules which are in Brownian movement (Mast, 
1926) and by the injection of a suspension of lamp black into it, the 
injected lamp-black particles spreading throughout this clear hyaline 
layer (Chambers, 1924). However, Schaeffer (1920) holds that some- 
times it is more rigid than endoplasm, and at other times not. 

Colloidal Nature of Protoplasm 

Any attempt to understand the processes occurring in protoplasm 
must depend for its success not only upon a knowledge of the chemical 
constitution of protoplasm but also of its physical structure. For this 
reason much ingenuity has been exercised in trying to discover the 
physical structure of the protoplasm in such relatively simple forms as 
Amoeba, in which permanently differentiated structures are at a mini- 
mum. For example, in A. proteus apparently only the nucleus is perman- 



50 PROTOPLASM OF PROTOZOA 

ently differentiated; the contractile vacuole and other structures are 
evanescent, at least in part. We have already mentioned the more ob- 
vious physical characteristics of Amoeba protoplasm, which consists, as 
does all protoplasm, of a complex heterogenous colloidal system, made 
up of a suspension (granular, fibrillar, and alveolar) of many different 
materials (fats, carbohydrates, and proteins) dispersed in a supporting 
continuous liquid part. The greatest volume ingredient in protoplasm 
is water; in solution in the water are various salts. Protoplasm may be 
deprived of many of its visible inclusions without killing it, leaving 
often a clear, colorless, optically homogenous hyaloplasm. Thus the 
microscopic structure of protoplasm gives no direct evidence of the 
finer submicroscopic structure. However, it is generally agreed that the 
finer structure of protoplasm is dependent upon its colloidal nature, 
which, because of the relatively large size of the particles present, results 
in enormous intracellular surfaces. 

Then too, the taking up of water by colloids is influenced both by the 
solutes present and by the previous history of the colloid itself (hys- 
teresis). Colloids often have the property of changing reversibly from 
a relatively liquid (sol) to a relatively solid condition (gel). A gel 
has many of the properties of a solid, among them elasticity, apparently 
due to its structure; but differs from a solid in that diffusion in gels of 
low concentration is often the same as in a simple solution, and in that 
chemical reactions often can occur at velocities unaffected by the gel 
condition. Every organism is dependent upon the temporal and spatial 
coordination of its chemical reactions, and this depends largely on the 
degree of dispersion and kinetic activity, because these regulate re- 
action velocities. Thus the organization of chemical events is due in 
some way to the nature and architecture of the colloidal system in which 
they occur. Therefore, biologists attempt to explain the physiological 
action of various factors as influences on the colloids of protoplasm. 

Consistency 

It is a phenomenon of general observation that protoplasm flows, 
but that it resists pressure. The former is a property commonly attributed 
to liquids, the latter to solids. In liquids there is great internal mobility 
of the molecules; this is essential to many physical activities, such as 



PROTOPLASM OF PROTOZOA 51 

movement, and to chemical reactions in protoplasm. On the other hand, 
solids have great internal cohesion of molecules, i.e., they retain their 
shape, show elasticity; this is essential to the maintenance of continuity 
and form of protoplasm. Colloids quite generally show changes from a 
relatively fluid to a firm jelly-like consistency, so that protoplasm as a 
colloidal system may partake of the nature of a solid (in the gel con- 
dition) or that of liquid (in the sol condition). Thus changes in vis- 
cosity are one of the essential factors in ameboid movement. In addi- 
tion, the viscosity of the protoplasm as a whole, and particularly the 
changes in viscosity within a given portion of a protozoan, are very 
important factors to be considered in connection with a study of mitosis, 
cytokinesis, cyclosis, rate of diffusion of various substances, protoplasmic 
reorganization, and functioning of such organs as the contractile vacuole. 

Besides the qualitative methods of estimating the viscosity of proto- 
plasm by observing the presence or absence of movement (Mast, 1926b) , 
presence or absence of Brownian movement (Bayliss, 1920; Pekarek, 
1930; Brinley, 1928), microdissection studies (Kite, 1913; C. V. Tay- 
lor, 1920; Chambers, 1924; Howland, 1924c; and others), certain 
experimental methods, i.e., centrifugation (Heilbrunn, 1928; 1929b; 
Fetter, 1926), electromagnetic methods (Seifriz, 1936), and rate of 
diffusion of certain dyestuffs (Chambers, 1924; Needham and Need- 
ham, 1926) have been used to reveal data as to the relative and absolute 
viscosity of the protoplasm of various Protozoa. For a detailed discus- 
sion of these various methods the reader is referred to the works of 
Chambers (1924), Heilbrunn (1928), and Seifriz (1936). 

The Protozoa show a wide range of viscosity values. In fact a varia- 
tion in protoplasmic viscosity from 2 times that of water in Amoeba 
(Heilbrunn, 1929b) to over 8,000 times that of water in Paramecium 
(Fetter, 1926) has been reported. Undoubtedly a much higher viscosity 
exists in many protozoan cysts and other forms characteristic of re- 
sistant stages. Seifriz (1936) has reported that during the winter the 
Plasmodium of a myxomycete becomes as hard and as brittle as a thin 
sheet of dry gelatin. 

In addition to the information obtained about the consistency of 
various Protozoa from multilation experiments, for example those of 
Calkins (1911) and Peebles (1912) on Paramecium caudatum, the 



52 PROTOPLASM OF PROTOZOA 

microdissection apparatus has proved to be a useful instrument in a 
study of the structure of Protozoa. By use of this method, Kite (1913) 
determined the ectoplasm of A. proteus to have a moderately high 
viscosity and cohesiveness. The substance forming the wall of the 
contractile vacuole seemed to possess a much higher viscosity than the 
surrounding endoplasm. The nucleus seemed to behave as a highly 
rigid granular gel. Kite further observed that the protoplasm of Para- 
mecium was a soft, elastic, and somewhat glutinous gel; the surface 
seemed to be more viscous than the interior. More recent studies by 
Chambers (1924); Rowland (1924a, 1924b); Rowland and Pollack 
(1927); Seifriz (1936), and others on various species of Amoeba 
have confirmed in a general way the observations of Kite. In addition, 
C. V. Taylor (1920, 1923) has described the pellicle of Euplotes patella 
as a firm, fairly tough, rigid substance. The micro- and macronuclei were 
found to be highly gelatinous, rather rigid structures imbedded in a 
viscous hyaline matrix. Needham and Needham (1926) in attempting 
to inject Opalina ranarum made the observation that the outer mem- 
brane is composed of a thick, tough substance; the inner cytoplasm they 
found to be jelly-like, so that injected indicators would not spread 
within it. The suggestion was tentatively made that the failure of the 
injected indicators to spread within the Opalina might be due to its 
consistency or to membranes surrounding the numerous nuclei. Follow- 
ing injection, the organisms continued to swim about, and in a few 
moments the injected portions suddenly dropped out, often leaving 
the animals quite riddled with holes. See also Chambers and Reznikoff 
(1926); Reznikoff (1926), and Morita and Chambers (1929), on in- 
jection experiments in Amoeba with similar results. 

Reilbrunn (1929b) has used the centrifuge method to study the 
absolute viscosity of A. dubia, which he found to be approximately 2 
times that of water at 18° C. Rowever, the viscosity was found to vary, 
with changes in temperature, from about 2 times water at 18° C. to 
25 times water at 21/2° C. (1929a). Pekarek (1930), by studying the 
Brownian movement in Amoeba, has estimated its viscosity to be about 
6 times that of water. More recently Seifriz (1936), by observing 
Brownian movement in Amoeba, estimated the viscosity in a quiet form 
to be 700 to 800 times that of water; in an active form it was much 
lower. Likewise Pantin (1924b) estimated the absolute viscosity of 



PROTOPLASM OF PROTOZOA 53 

certain marine amoebae to be comparable to that of vaseline, i.e., over 
1,000 times that of water. 

Fetter (1926), using the same method as Heilbrunn, found the 
absolute viscosity of Paramecium to be 8,027 to 8,726 times that of 
water. Hyman (1917) has found that the heliozoon Actinosphaerium 
eichhornii can be cut into pieces as if it were solid. In addition to the 
viscosity and the changes in viscosity of the protoplasm which may take 
place within organisms under "normal" conditions, certain experi- 
mental conditions, such as abnormal salt concentration, acids, and 
alkalies, abnormally high or low temperature, mechanical agitation, 
changes in hydrogen-ion concentration, anesthetics and narcotics, radia- 
tion, sound waves, and so forth, may cause marked changes in the 
viscosity of the protoplasm. 

The effect of various agents (chemical, mechanical, electrical, and 
so forth) upon the viscosity of protozoan protoplasm has been deter- 
mined by studying their effect upon locomotion, Brownian movement, 
body form, pseudopod formation, rate of action of the contractile 
vacuole, as well as by centrifuging and by the microscopic appearance 
of the cytoplasm. 

THE EFFECTS OF WATER 

Since the principle solvent of protoplasm is water, any condition 
which tends to increase or decrease the water content of the organism 
also tends to change the viscosity or consistency of its protoplasm. Thus 
hypertonic solutions and desiccation usually cause the cells to shrink, 
this being accompanied by an increase in the viscosity of their proto- 
plasm; a hypotonic solution or the injection of water directly into the 
organism tends to induce a swelling, accompanied by a decrease in the 
viscosity of the protoplasm (see Pantin, 1923) . 

THE EFFECTS OF SALTS 

The effects of salts on the consistency of certain Protozoa, particularly 
Amoeba, have been rather extensively studied. Giersberg (1922), Ed- 
wards (1923), Chambers and Reznikoff (1926), Reznikoff and Cham- 
bers (1927), Pantin (1926a, 1926b), Brinley (1928), Heilbrunn and 
Daugherty (1931, 1932, 1933, 1934), Thornton (1932, 1935), Pitts 
and Mast (1934), Butts (1935), and others have studied the action 



54 PROTOPLASM OF PROTOZOA 

of various salts and salt antagonisms upon the consistency of Amoeba 
protoplasm. In general it has been found that sodium and potassium 
ions tend to increase the viscosity of the internal protoplasm, while 
calcium and magnesium tend to lower it. However, Heilbrunn and 
Daugherty (1932, 1934), have found that this does not hold for the 
plasmagel (i.e., outer layer) of A. proteus. Here calcium produces a 
pronounced stiffening of the cortical gel, and this effect tends to be 
antagonized by Na, K, and Mg. Potassium has the strongest liquefying 
effect, Mg next, and Na the least action. The degree of reaction, par- 
ticularly the antagonism of salts, seems to vary somewhat, depending 
upon the hydrogen-ion concentration (Pitts and Mast, 1934). 

Greeley (1904) observed that KCl coagulates or increases the 
viscosity, whereas NaCl liquefies or decreases the viscosity of the proto- 
plasm of Paramecium. Heilbrunn (1928) reports that unpublished 
work of Barth shows that lithium salts cause a coagulation or increase 
in viscosity of the protoplasm of both Stentor and Paramecium. Heil- 
brunn (1928) found that sodium, potassium, ammonium, and lithium 
chlorides all cause coagulation of Stentor protoplasm and that weak 
solutions of HgClg produce a coagulation of the protoplasm of Euglena. 
The effect of salts upon reproduction in Amoeba has been studied by 
Voegtlin and Chalkley (1935) and by Butts (1935). Oliphant (1938) 
observed that potassium, lithium, sodium, and ammonium salts induce 
reversal in the direction of the effective beat of the cilia of Paramecium, 
whereas calcium and magnesium do not. The reversal in direction of the 
beat of the cilia is thought to be associated with an increase in viscosity 
of the cytoplasm. See also the work of Spek, 1921, 1923, and 1924, 
on the action of various salts on Actinosphaerium, Opalina, and other 
Protozoa. 

Chambers and Howland (1930) have cut or torn Spirostomum 
in CaCl, solutions; the exposed protoplasm coagulates into a dense mass 
which the uninjured part of the organism pinches off. Injection of 
CaClg produces localized coagulated regions which are pinched off. 
Potassium chloride and NaCl cause liquefaction. Ephrussi and Rap- 
kin (1928), however, report that CaClg facilitates "I'explosion" of this 
ciliate; KCl and NaCl render explosion more difficult. 

Chambers and Howland (1930) have further performed injection 
and immersion experiments with A. eichhornii, a heliozoon with grossly 



PROTOPLASM OF PROTOZOA 55 

vacuolated protoplasm. Immersion in NaCl or KCl dissolves the vacuolar 
membranes, with a dissolution of the intervacuolar protoplasm. Im- 
mersion in strong concentrations of CaCL causes coagulation of the 
protoplasm; in weak solutions the coagulation may be local, and the 
living remnant rids itself of the coagulated regions. After immersion 
in strong concentrations of MgCL the protoplasm coagulates into a 
flabby mass; in weaker solutions localized regions rupture. Injections 
result in similar but more localized effects, except in the case of MgClg. 

THE EFFECTS OF ACIDS AND ALKALIES 

There seem to be no general agreement on the effects of acids upon 
protozoan protoplasm. Some authors report that they cause an increase 
in viscosity, while others find that they produce a decrease in viscosity 
of the protoplasm. 

Jacobs (1922), by bubbling CO, through the culture medium con- 
taining Paramecium and Colpidium, observed that short exposure of 
these organisms to the CO, caused a decrease in the viscosity of the proto- 
plasm, while longer exposures increased it. Brinley (1928) found that 
CO2 caused gelation of the ectoplasm and solation of the endoplasm of 
A. proteus. Reznikoff and Chambers (1927), after injecting bubbles of 
CO2 into A. dubia, observed that it produced a decrease in the viscosity 
of the protoplasm and that the animal was not irreversibly injured un- 
less the CO2 destroyed the cell membrane. Hydrochloric acid has been 
observed to produce an increase in the viscosity of the protoplasm of 
Amoeba (Chambers, 1921; Edwards, 1923; Brinley, 1928). Heilbrunn 
(1937) finds that acids cause an increase in viscosity of both the plasma- 
sol and plasmagel of Amoeba. 

Chambers (1921) reports that basic dyes, which contain a relatively 
strong acid radical, jelly the protoplasm of Amoeba, whereas acid dyes, 
with a strong basic radical, liquefy it. 

The action of alkalies has been reported to decrease the viscosity of 
A. proteus (Chambers, 1921; Edwards, 1923; Brinley, 1928). How- 
ever, Heilbrunn (1937) reports that alkalies increase the viscosity of 
the plasmasol and decrease the viscosity of the plasmagel in Amoeba. 

THE EFFECT OF TEMPERATURE 

As pointed out by Brues (1927), the Protozoa are among the most 
resistant of all animals to high temperatures; they have been found liv- 



56 PROTOPLASM OF PROTOZOA 

ing in hot springs at temperatures between 50° and 60° C. Motile forms 
seem to be much more susceptible to high temperature than do en- 
cysted forms (cysts of Colpoda are not killed at 100° C. dry heat for 
three days), a condition probably associated with the low water con- 
tent of the organisms (Bodine, 1923). That Protozoa show ability 
gradually to acclimate themselves to increased temperatures has been 
shown by Jacobs (1919). 

Since temperature changes are known to affect the rate of action of 
molecules in liquids, it is only reasonable to assume that their effect is 
somewhat similar upon protoplasm. In general it may be stated that a 
slight increase of temperature over that of the normal, causes a decrease in 
protoplasmic viscosity; a slightly higher temperature, a reversible co- 
agulation, and a still higher temperature, an irreversible coagulation 
and death. Davenport (1897) has referred to these conditions as con- 
traction, heat rigor, and death rigor respectively. See also the discus- 
sion of the action of temperature on protoplasm by Heilbrunn, 1928. 

Pantin (1924a), working on two species of marine Amoeba, found 
that the viscosity was high near 0° C. and decreased with rise in tempera- 
ture. The primary effect of temperature upon locomotion, he holds to be 
a direct effect upon the change of state of the protoplasmic sol-gel trans- 
formation. 

Perhaps one of the most complete studies of the action of temperature 
upon protozoan protoplasm is that of Heilbrunn (1929a) on A. dubia. 
He found that at temperatures from about 3° to 10° C, the viscosity 
value was about 22 to 23 times that of water. From 10° to 18° C. there 
was a very rapid decrease in viscosity, approaching about 2 to 3 times 
that of water, while at 20° to 25° C. there was an increase in viscosity to 
about 8 to 9 times that of water, followed by a decrease to about 2 times 
that of water at 30° to 35° C. Although the range of Heilbrunn's experi- 
ments was not extended beyond 35° C, he states that higher tempera- 
tures cause a coagulation of the protoplasm. Thornton (1932) has 
observed that the maximum viscosity of the plasmagel of A. proteus 
occurs at 4.5° C. Between 4.5° and 30° C. the viscosity decreases pro- 
gressively, with rise in temperature, until at 30° C. the decrease is more 
rapid. Thornton (1935) has further found that the action of certain 
salts does not alter the fundamental effect of temperature on the vis- 
cosity of the plasmagel of A. proteus. Daniel and Chalkley (1932) ob- 



PROTOPLASM OF PROTOZOA 57 

served that the rate of mitosis, including nuclear and cytoplasmic divi- 
sion, of A. proteus varies inversely with temperature from about 4° to 
30° C. From about 30° to 40° C. these processes vary directly with the 
temperature. 

Greeley (1904) found that the consistency of the protoplasm of vari- 
ous Protozoa, like organic colloids, varies directly with the temperature, 
within certain limits. As the temperature is elevated above the normal, 
the protoplasm absorbs water, so that its fluidity and motility are greatly 
increased. These changes continue until a critical point is reached, at 
which coagulation occurs. The resistance of P. caudatum to a tempera- 
ture of 40° C. has been shown to vary with the hydrogen-ion concentra- 
tion of the medium and to exhibit two maxima of resistance: one on 
the alkaline and one on the acid side, with a region of minimal resist- 
ance at neutrality (Chalkley, 1930). In saline solutions the mechanism 
of death by heat seems to vary with different hydrogen-ion concentra- 
tions. At pW. 6 or less the cell coagulates, at pH 8 or more the organism 
disintegrates; between these two extremes death occurs from rupture by 
swelling. Furthermore, Chalkley also found an increase in thermal re- 
sistance of Paramecium on the addition of Ca, and a decrease on addition 
of K. Oliphant (1938) found that the rate at which the cilia of Para- 
mecium beat in reverse varies directly with temperature, a condition he 
implies is associated with changes in viscosity of the organisms. For a 
further discussion of the effects of high temperatures on organisms, in- 
cluding the Protozoa, see Belehradek (1935). 

It has been shown by Greeley (1901) that when Stentor coeruleus is 
suddenly lowered to the freezing point of water, it is usually killed. 
However, when the temperature is lowered slowly to 0° C., a remark- 
able dedifferentiation of the animal takes place. The resorption of the 
cilia and the gullet, and the throwing off of the ectosarc was observed, 
and there was finally formed a spherical cyst-like undifferentiated cell, 
which Greeley referred to as a "resting" cell. When returned to room 
temperature, a reverse process takes place, and the cyst-like organism 
becomes active. With the lowering of the temperature the organism was 
observed to lose water. Greeley (1903) further found that the method 
of reproduction in Monas could be controlled by temperature; at 20° C. 
the organisms reproduced sexually and by fission; at from 1° to 4° C. 
they reproduced by asexual spores. 



58 PROTOPLASM OF PROTOZOA 

According to Luyet and Gehenio (1938), Becquerel (1936) found 
that certain Amoeba in dry soil were not killed when subjected to 
— 269° to — 271° C. for 7.5 days, or when subjected to ^190° C. for 
480 hours. See the above-mentioned work of Heilbrunn (1929a) for 
absolute viscosity values of the protoplasm of A. duhia at low tempera- 
ture. 

Chambers and Hale (1932) observed that Amoeba exposed to — 5° 
C. were not killed. However, by inserting an ice- tipped pipette into the 
interior of the organism, fine feathery crystals of ice were observed to 
appear immediately at — 0.6° C. 

In general it may be stated that low temperatures tend to increase the 
viscosity and thus to decrease the rate of locomotion, and to favor cyst 
formation in Protozoa. Motile forms are usually killed as the tempera- 
ture of the water reaches 0° C. However, the Protozoa show some degree 
of acclimatization to low temperatures. 

MECHANICAL AGITATION 

Mechanical agitation may cause a marked change in the consistency 
of Amoeba protoplasm, apparently by causing a breakdown (thixotropic 
collapse) of its internal structure. Chambers (1921) has shown that 
churning Amoeba by microneedles caused a liquefaction of its proto- 
plasm. Vigorous shaking (Angerer, 1936) caused at first a liquefaction 
of the plasmasol of A. dub'ia, followed by an increase in viscosity; con- 
tinued agitation caused the complete dissolution of the organism. How- 
ever, in A. pro tens agitation caused a decrease in viscosity in the plasma- 
gel to a minimum, without the subsequent increase observed in the 
plasmasol of A. dub'ia. High-frequency sound waves have been observed 
to produce whirling of the inclusions in the small vacuoles of A. proteus 
and A. dubia; higher intensities cause a mild whirling of the more liquid 
regions, followed by rupture of the organisms (E. N. Harvey, E. B. 
Harvey, and Loomis, 1928). A decrease in viscosity of the endoplasm 
was also observed. 

HYDROGEN-ION CONCENTRATION 

That Protozoa can live in wide ranges of hydrogen-ion concentrations 
is evident from the work of Alexander (1931) on Euglena which he 



PROTOPLASM OF PROTOZOA ^9 

found lived in ranges from pH 2.3 to pH 11. Studies on the effects of 
hydrogen-ion concentration on protoplasmic viscosity are complicated by 
the rate of entrance of the ions and by the fact that they may be neutral- 
ized by the buffers of the protoplasm (Pollack, 1928a) . 

Pantin (1926a) has found that the movement of certain marine 
amoebae takes place between /;H 6 and 10. It is reversibly inhibited at 
the acid limit, but alkaline inhibition is reversible only after a brief Im- 
mersion. The rate of movement he holds to depend upon the rate of 
change in s6l:;:±gel transformation. In addition, the water content of 
A. p rote us has been shown to vary with the hydrogen-ion concentration 
of the medium (Chalkley, 1929). The effect of changes in hydrogen-ion 
concentration upon the action of cilia has been studied by Chase and 
Glaser (1930). 

THE EFFECTS OF NARCOTICS 

For a summary of the literature on the effects of narcotics in cells gen- 
erally, the reader is referred to the work of Winterstein (1926) and of 
Henderson (1930). In general it may be stated that the action of nar- 
cotics upon cells is to change their permeability and viscosity, to inhibit 
enzyme action, and to affect the electric potential. 

Alcohol has been reported to produce a lowering of the viscosity of 
the protoplasm of A. proteus (Edwards, 1923; Brinley, 1928). Ether 
and chloroform likewise have similar effects (Brinley, 1928). Certain 
paraffin oils have also been reported to have a narcotic effect on A. dubia 
(Marsland, 1933). More recently Daugherty (1937) found that the 
higher alcohols and ether in concentrations just below lethal cause lique- 
faction of the plasmagel of Amoeba; the same concentrations of the 
lower alcohols produce gelatin of the plasmasol; the higher alcohols and 
ether, liquefaction of the plasmasol. More dilute solutions of the higher 
alcohols and ether produce first liquefaction and then gelatin of the 
plasmasol (see also Frederikse, 1933a). Potassium salts and fat solvents 
liquefy the plasmagel in A. proteus (Heilbrunn, 1931). 

Makarov (1935) has studied the effects of narcotics on various Infu- 
soria, using vital stains in conjunction with the ultramicroscope. Narcotics 
cause a change in the dispersion of the colloids, which is reversible. 
Strong concentrations cause an irreversible coagulation. 



60 PROTOPLASM OF ^ROTOZOA 

THE EFFECTS OF RADIATION 

The Specific effects of radiation upon the cell are unknown. Further- 
more, whether the nucleus or the cytoplasm is more susceptible to radia- 
tion is a debated question. According to Heilbrunn and Mazia (1936), 
Glocker and Reuss claim that isolated cells are less sensitive to Roentgen 
rays than are cells in mass. 

The permeability of 'Paramecium and Stylojiychia to NH^OH has been 
shown to increase with increased exposure of the animals to radiation 
(Packard, 1923, 1924). After reviewing the literature on the biological 
effects of radiation, including numerous studies on Protozoa, Heilbrunn 
and Mazia (1936) reach the conclusion that ultra-violet rays, Roentgen 
rays, and radium all cause liquefaction of the protoplasm and, with an 
increase in exposure, coagulation. Coagulation of the protoplasm is fre- 
quently preceded by extensive vacuolization. Furthermore, Heilbrunn 
and Daugherty (1933), from their work on the effects of ultra-violet 
rays on Amoeba, offer the theory that the effect of radiation is to release 
the bound calcium from the cell cortex; it then enters the endoplasm, 
causing first liquefaction and then gelation. Likewise, ultra-violet radia- 
tion causes a release of fat in Amoeba (Heilbrunn and Daugherty, 
1938). For the recent ingenius method of "microdissection" with ultra- 
violet rays, see Tchakhotine (1937). 

THE EFFECT OF HEAVY WATER 

E. N. Harvey (1934) has studied the effects of heavy water on Para- 
mecium, Amoeba, Euglena, and Epistylis (see also Taylor, Swingle, 
Eyring, and Frost, 1933). Harvey found that paramecia were killed by 
80 to 100-percent heavy water in from 6 to 10 hours. They first swim 
slowly, appearing bloated; the contractile vacuole stops functioning; blis- 
ters appear, followed by disintegration. However, they were not markedly 
affected by 0.2-percent heavy water. Amoeba rounds up and is killed in 
from 4 to 6 hours in 80 to 100-percent heavy water. Euglena, on the 
other hand, is not irreversibly injured by 90 to 97-percent heavy water. 
Gaw (1936b) found Blepharisma to become more spherical in 95-per- 
cent heavy water, which indicates a change (decrease in viscosity) in the 
physical nature of its cytoplasm. 



PROTOPLASM OF PROTOZOA 61 

HYDROSTATIC PRESSURE 

High hydrostatic pressure (500 atmospheres) causes collapse of 
pseudopodia, and a rounding up o( A. proteus (Brown and Marsland, 
1936). This effect is apparently the result of liquefaction of the plasma- 
gel, an effect which inhibits the normal sol-gel transformation. However, 
similar high hydrostatic pressures seemed to have little or no effect on the 
beating of flagella or cilia of other Protozoa (Marsland, 1939). 

THE EFFECTS OF ELECTRIC CURRENT 

The effects of electric current on many Protozoa have been studied 
(see Hahnert, 1932, for literature). 

Bayliss (1920) has shown that on passage of an electric current of 
the proper intensity through Amoeba, immediate gelation of the proto- 
plasm takes place and all Brownian movement stops. As the Amoeba re- 
covers, the protoplasm solates and the particles again take up their active 
motion. Strong electric shocks caused irreversible coagulation. More re- 
cently Luce (1926), Mast (1931b), and Hahnert (1932) have studied 
the effects of electric current on Amoeba. In general, as stated by Hah- 
nert, a constant electric current provokes responses in Amoeba by defi- 
nite polar actions at the ends of the organism. Destruction or solation 
of the plasmagel occurs at the cathode end immediately, then contractive, 
and later, disintegrative processes occur at the anodal end. These re- 
sponses are directly dependent upon the strength of the current. 

IRREVERSIBLE COAGULATION 

Acids and certain salts, such as mercuric chloride, in the proper con- 
centrations, cause irreversible coagulation of protoplasm. Protozoa are 
therefore often "fixed" in such solutions for morphological studies. The 
causes of the irreversible coagulation which occurs at "normal" death 
are obscure. 

Surface Properties 

A discussion of the physical properties of the surface layer in Protozoa 
involves its structure, which has been analyzed by a study of its gross 
morphology, elasticity, contractility, extensibility, viscosity, and ultra- 
microscopic architecture. It is obvious that any conception of the physi- 
cal structure of the surface membrane must be in harmony with its 



62 PROTOPLASM OF PROTOZOA 

functions, such as preserving the integrity of the organism (by being 
immiscible with water), controlhng the diffusion of materials in and out 
of the cell (permeability), acting as a seat of electromotive forces (mem- 
brane or diffusion potentials), maintaining form (possessing tension), 
forming secretions (for protection, adhesion, forming cyst walls, armor, 
and so forth) and for the reception of stimuli, and so on. 

STRUCTURE AND ORIGIN 

The problem of surface structure in the Protozoa is often complicated 
by a failure of many investigators to define clearly what they refer to as 
the "cell membrane." From a review of the literature it is quite obvious 
that the vital membrane essential to the cell may be reduced to only 
a very thin ultramicroscopic film, such as that which forms at the torn 
surface of an amoeba. In other cases the "cell membrane" is of micro- 
scopic dimensions and displays the physical properties of a thick, tough 
pellicle. In this connection the work of Nadler (1929) showed that the 
pellicle could be completely removed from Blepharisma without killing 
or even affecting the shape of the organism; after a few days a new pel- 
licle may be formed in these animals. Could it not be, therefore, that in 
forms with thick surface layers, the physiologically active vital mem- 
brane is mainly limited to a thin film, and that the remaining part of 
the thick surface layer serves mainly for protection, support, giving an- 
chorage to locomotor organs, secretion of slime for adhesion, forming 
cyst walls, or in the formation of other surface structures which possibly 
aid the animal in coping with its environment? 

In addition to the vast literature describing definite morphological 
membranes at the surface of various Protozoa, both in the living and 
fixed condition, considerable experimental work has been done on the 
physical nature of the surface layers. Kite (1913), Chambers (1924), 
Howland (1924c), Rowland and Pollack (1927), Taylor (1920), 
Needham and Needham (1925, 1926), and many others have actually 
established the presence of the surface membrane by puncturing it, tear- 
ing it, and in some cases actually removing it. In general it has been 
found to vary in thickness from that of a delicate film to that of a tough 
pellicle. Microdissection methods have also shown it to possess measur- 
able elasticity and contractility, and to vary considerably in consistency 
from that of the underlying protoplasm. 



PROTOPLASM OF PROTOZOA 63 

The tension at the surface of Amoeba has been measured by E. N. 
Harvey and Marsland (1932). They injected drops of paraffin oil or 
oHve oil into A. proteus and A. duhia and then subjected them to centri- 
fugal force in the microscope-centrifuge. Because of the bouyancy of the 
oil, the organisms became stretched. The amount of distortion was photo- 
graphed and, under certain assumptions, a value for the order of mag- 
nitude of the surface forces was calculated. In this way these authors 
found the tension at the surface of A. duhia to give values of one to three 
dynes per centimeter. In this form they concluded that "there can be no 
appreciable turgidity due to resisting surface layers." However, in A. 
proteus it was impossible to pull even large oil droplets out of the or- 
ganism by the highest centrifugal force available. For this reason no 
tension at the surface of this organism could be calculated, but they esti- 
mated it to be about thirty times that for A. duhia. The surface of A. pro- 
teus was described as a firm, tough, external layer. 

Since the surface membrane may be of ultramicroscopic dimensions, 
its physical properties are not easily determined, and for this reason 
much of our knowledge is of a theoretical nature. It has been held that 
the appearance of a new membrane at the surface of a torn bit of proto- 
plasm is due to the accumulation at the surface of substances, chiefly 
lipoid, which tend to lower surface tension. However, Heilbrunn thinks 
of this process of new surface membrane formation as a "surface precipi- 
tation reaction," comparable in many ways to the clotting of blood. He 
has produced evidence to show that the presence of calcium is a pre- 
requisite for the formation of new surface membranes. In addition, he 
holds that any factors which cause a release of calcium from its protein 
binding cause the "surface precipitation reaction" to take place within 
the cell interior, giving to it a froth-like appearance. 

Whatever may be the exact mechanism of new membrane formation, 
the fact that a time factor is involved in its production from the cyto- 
plasm, that it assumes increased tension over that of the underlying 
cytoplasm, that its consistency and durability depend both upon the en- 
vironmental medium and the specific character of the protoplasm from 
which it is formed, as well as its semipermeable properties, all point to 
the surface membrane as being a definite, organized structure (Cham- 
bers, 1924). 

Recent researches upon the optical properties of cell membranes other 



64 PROTOPLASM OF PROTOZOA 

than those of Protozoa, by means of polarized Hght and X-ray diffrac- 
tion methods, seem to indicate that they are constructed of hpoid and 
protein molecules, with their long axes arranged perpendicular and 
parallel, respectively, to the surface of the cell (Schmitt, 1938). E. N. 
Harvey and Danielli (1936) also hold the cell membrane to be com- 
posed of lipoid and protein substances. Furthermore, Langmuir has 
shown that the structure and surface properties of certain nonliving 
membranes are frequently determined by the orientation of the indi- 
vidual molecular layers; these may, under certain conditions, undergo 
almost instantaneous reversal or reorientation. This overturning may 
markedly alter the chemicophysical properties of the membrane, a fact 
which may prove to be of considerable interest in the study of the 
permeability of cells generally. As pointed out by Harvey (1936), one 
would seem justified in conceiving of the surface membrane in different 
cells as composed of a monomolecular layer and a polymolecular film, or 
as a polymolecular oil film with oriented adsorbed protein molecules 
varying from a rather liquid to solid consistency. 

The remarkable film-forming properties of the ciliate Spirostomum 
have been studied by Faure-Fremiet, Ephrussi, and Rapkine (1926). 
This organism explodes when it comes into contact with the air-water 
interface, and its solid constituents spread over the surface of the water. 
By first dusting the water with talc, it was determined that the surface 
film was 4.2 to 5.7 |j p thick, and probably monomolecular. 

PERMEABILITY 

1. Cell membrane. — In Protozoa as in all other types of cells one of 
the most important properties of the protoplasm is its ability to form 
new surface membranes which have a selective permeability. In addition 
to preserving the integrity of the cell, the surface membrane regulates 
to a large extent the passage of dissolved foodstuffs and oxygen into the 
cell and the diffusion of waste materials from it. The rate of this exchange 
depends in part upon the degree of the permeability of the surface mem- 
brane and in part upon the osmotic concentration of the cell and of the 
surrounding medium. 

It is not our purpose to discuss generally here the physiological prob- 
lems of permeability and diffusion. However, many fresh-water Proto- 



PROTOPLASM OF PROTOZOA 65 

2oa possess an interesting mechanism for controlling their osmotic state. 
The protoplasm of these organisms possesses a much higher osmotic 
concentration than that of the surrounding medium. For this reason 
water tends continually to enter them, and, were it not for the controlling 
mechanism of the surface membrane and the continuous bailing out 
process of the contractile vacuole, the animals would swell up and burst. 
Adolph ( 1931 ) has reviewed the literature dealing with the rate of water 
exchange in Protozoa and has found that the fastest turnover is in 
CryptochUum, which excretes its body volume in 2 minutes; the slowest 
water exchange was in Amoeba, which requires 31.5 hours, while P. 
caudatum eliminates its own body volume of water in from 15 to 
20 minutes. 

Water, when injected in small amounts into an amoeba, readily dif- 
fuses throughout the cytoplasm, causing a temporary cessation of ame- 
boid movement (Chambers, 1924). However, if the injection be great 
in amount i.e., equal to half the body volume, the water tends to collect 
on one side in the form of a large blister, which is eventually pinched off, 
and within a short time the amoeba resumes its normal activities (How- 
land and Pollack, 1927). In addition, a marked increase in contraction 
rate and water output of the contractile vacuole was noticed. 

In contrast to the above experiment. Mast and Doyle (1934) caused 
A. proteus, A. duhia, A. radio sa, and A. dofleini to lose water by plac- 
ing them in 3-percent egg albumen, hypertonic salt solutions, or solutions 
of calcium gluconate. The organisms decreased in size and their surfaces 
became wrinkled and covered with protuberances, folds, and crevices. 
The crests of some of the adjoining protuberances and folds fuse, giv- 
ing rise to tubes open at one end. The region of the fused folds push 
out, forming a pseudopodium, thus extending the tubule, the mouth of 
which was observed to expand and contract, drawing the fluid in. Later 
the tubule disintegrates, releasing the fluid to the inside of the organ- 
ism. This "drinking" of water by means of tubules seems to be an im- 
portant mechanism, to compensate for the rapid loss of water in the 
organism. Further experiments by Mast and Fowler (1935) showed that 
when A. proteus was placed in Ringer solution containing various con- 
centrations of lactose, it would decrease as much as 88 percent in total 
volume without injury. The rate at which the water leaves the cell was 



66 PROTOPLASM OF PROTOZOA 

found to be approximately 0.026 cubic micra per minute through each 
square micron of surface. These authors concluded that the permeability 
to water was regulated by the plasmalemma. 

It is well known that many marine Protozoa, especially Sarcodina, do 
not possess contractile vacuoles, but, when transferred to fresh water, 
contractile vacuoles may appear (Schaeffer, 1926). The contractile vacu- 
oles of fresh-water forms either work very slowly or disappear entirely 
upon the organism being transferred to various concentrations of sea 
water. Furthermore, some Protozoa of the same species, for example 
Actinophrys, may be found in both fresh and salt water. The cytoplasm 
of the fresh-water form is greatly vacuolated and possesses a contractile 
vacuole, while the cytoplasm of the salt-water form is relatively free of 
vacuoles, including the contractile vacuole, and the general appearance 
of the cytoplasm has changed to a granular condition. Thus a gradual 
acclimatization of fresh-water Protozoa to salt water seems to reduce the 
difference between the external and internal osmotic pressures by a loss 
of water from the cell. This is probably accompanied by changes in the 
physical state of the protoplasm, particularly its consistency and specific 
gravity. It is of interest to recall here the extraordinary case of Noctiluca, 
in which the specific gravity of the organism is less than that of the sur- 
rounding sea water, owing to lower concentration of salts. Here the 
water, instead of entering the organism, tends to diffuse out of it, so 
that osmotic work must be done to retain its constant state. 

Experiments designed to test the selective permeability of the surface 
membrane of the Protozoa have been carried out by utilizing various 
dyestuffs (Chambers, 1922; Ball, 1927). For example, according to 
Chambers (1922) an aqueous solution of eosin does not stain Amoeba 
from the exterior. However, if injected into the interior of the cell, it 
readily diffuses throughout the protoplasm. 

Attempts to visualize the mechanism of permeability control in sur- 
face membranes have been made by assuming a solubility of the permeat- 
ing substance in the membrane inself or by assuming the membrane to 
have a sieve-like structure similar to a filter, but much smaller. In any 
case, whatever may prove to be the final answer to this problem, it will 
undoubtedly involve a change in molecular aggregation, organization, 
and polarity of the elements of the physiological membrane. 

2. Nuclear memhrane. — The interphase nucleus, like the cytoplasm of 



PROTOPLASM OF PROTOZOA 67 

the Protozoa, is surrounded by a limiting surface membrane which may 
be demonstrated in many forms by the usual cytological techniques (see, 
for instance, Chalkley, 1936). In addition, experimental studies on the 
living nucleus by Kite (1913), Chambers (1924), C. V. Taylor (1920), 
and many others have substantiated the view of the presence of a defi- 
nite nuclear membrane, often extremely thin, but usually composed of a 
moderately tough, solid substance, which, upon rupture of the surface 
of the organism, may preserve the integrity of the nucleus for a consider- 
able time. Seifriz (1936) reports that the nuclear membrane of Amoeba 
may be readily removed by microneedles, following coagulation of the 
nucleus as a whole. 

Morita and Chambers (1929) have shown that the nuclear membrane 
of Amoeba is permeable to acid, while the general body surface of the 
animal is not. 

King and Beams (1937) report that the macronucleus of F. caudatum 
in the vegetative stage was greatly stretched by centrifugation. In some 
specimens it was separated into a relatively heavy chromatic portion and 
a relatively hght achromatic portion. Animals with their macronucleus 
separated in this way were able to live and carry on apparently normal 
metabolic processes. Here it is evident that a mechanical disruption of 
the macronucleus did not cause a marked physical change of the cyto- 
plasm, as often happens when the nucleus is punctured or cut by a needle 
(Kite, 1913; Peebles, 1912). King and Beams were unable to differenti- 
ate a limiting membrane surrounding the two separated portions of the 
macronucleus, and, if present, it must have been of ultramicroscopic di- 
mensions. 

Luyet and Gehenio (1935) were unable to demonstrate a definite 
membrane surrounding the macronucleus of P. caudatum by means of 
ultra-violet absorption methods. 

Whatever, in the final analysis, the physical structure of such ultra- 
microscopic nuclear membranes may prove to be, it will probably in- 
volve a special molecular behavior, characteristic of surfaces much like 
that which is thought to occur between nonliving immiscible fluids. 

3. Contractile vacuole. — For the early literature dealing with the long- 
disputed question of the presence or absence of a permanent membrane 
surrounding the contractile vacuole in Protozoa, the reader is referred to 
the works of C. V. Taylor (1923), Lloyd (1928), Howland (1924a), 



68 PROTOPLASM OF PROTOZOA 

King ( 1935 ) , and Kitching ( 1938) . It is well known that the contractile 
vacuole often arises from the coalescence of smaller vacuoles. 

C. V. Taylor (1923) studied the structure of the contractile vacuole 
of Euplotes and found that it appeared to be composed of a definite 
"wall" of measurable thickness. However, he later concluded that the 
apparent "wall" was an optical illusion and that careful study revealed 
only the internal surface of the "wall" to be sharply delimited; externally 
it merges not abruptly, but gradually, into the surrounding medium. By 
moving the needle point against and about the contractile vacuole, he 
found it could be displaced and that its boundary was tolerably durable 
and its viscosity distinctly higher than the surrounding endoplasm. With 
completion of contraction, the vacuole wholly disappears. 

In a similar study upon the contractile vacuole of A. verrucosa and P. 
caudatum, Rowland (1924b) and Howland and Pollack (1927) have 
been able to dissect the contractile vacuole out of the organisms and ob- 
serve it floating freely in the water. Here it may be stained by alizarin 
blue and manipulated by microneedles. Upon puncture, the surface of 
the membrane was observed to wrinkle. In other experiments, when the 
contractile vacuole was forced into contact with the plasmalemma, a 
fusion took place and, because of this fact, Howland and Pollack were 
led to suggest that the surface membranes of both the cell and the con- 
tractile vacuole must possess similar physical properties. These authors 
further found the contractile vacuole to lie in a region of gelated endo- 
plasm, a condition they think necessary for its functioning. 

Mast (1938) has described the membrane at the surface of the con- 
tractile vacuole in A. proteus as a well-differentiated structure about 
0.5 micron in thickness. 

King (1935) has described the permanent components of the contrac- 
tile vacuole system of P. mtdthnhronucleata as including the pore with 
its discharging tubule, and the feeding canals, each made up of a distal 
excretory portion, an ampulla, and an injection tubule. The membrane 
of the contractile vacuole itself is considered a temporary structure, dis- 
appearing at systole and closing the pore, which ruptures at the next 
systole. The new membrane of the contractile vacuole appears by the 
coalescence of the membranes of vesicles which lie just under the pore, 
and becomes continuous with that of the pore. In centrifuged P. cauda- 
tum, King and Beams (1937) observed that in some cases the vacuole 



PROTOPLASM OF PROTOZOA 69 

seems to have been moved out of its position in contact with the mem- 
brane closing the pore. In spite of this, the feeding canals continue to 
form other main vacuoles, each of which expels its contents through 
the pore in normal fashion. The displaced vacuole is free in the endo- 
plasm, and, if not too large, may move about in the protoplasmic stream. 
Such vacuoles have been observed to be present in the cytoplasm twenty- 
four hours after centrifuging. These observations were interpreted in 
further support of the view that the main contractile vacuole and its 
membranes are purely temporary, forming anew before each systole by 
the fusion of feeding vesicles formed at the vacuolar ends of the feeding 
canals. The feeding canals are markedly osmiophilic, and for this reason 
they have been described by Nassonov (1924) as homologous to the 
Golgi apparatus. However, this view is not supported by Beams and 
King (1932) and King (1935). Gelei (1928) thinks of the cytoplasm 
surrounding the feeding canals as "nephridial-plasma" and suggests a 
parallelism between the nephridial system in Paramecium and that of 
higher organisms. Metcalf (1910) observed, in the cytoplasm surround- 
ing the contractile vacuole of A. proteus, small round granules which 
he termed "excretory granules." These bodies, which are permanent 
structures, are thought by Metcalf to be functionally connected with 
secretion. However, Mast (1938a) has been unable to substantiate this 
view. 

It is interesting to note that the permeability of the cell membrane and 
that of the contractile vacuole system are in some ways similar. For in- 
stance, Morita and Chambers (1929) report that in A. duh'ia both the 
surface membrane and the contractile vacuole membrane are imperme- 
able to HCl. Kitching (1936) reports that the vacuolar surface, like 
the cell surface, is relatively impermeable to salts in the peritrich ciliates. 
He further presents arguments to show that the vacuolar system in these 
forms actively secretes water. 

4. Vood vacuoles. — In many free-living Protozoa the ingested food 
particles are surrounded by a distinct vacuole. It is here that the digestive 
enzymes collect and act upon the solid food particles, converting them 
into a dissolved form suitable for use by the organism. The so-called food 
vacuolar membranes, in forms such as Amoeba and ParajJiecium, must 
perform much the same function as the cell membranes surrounding the 
cells in the intestine of higher organisms. In other words, the semiperme- 



70 PROTOPLASM OF PROTOZOA 

able membrane delimiting the food particles from the surrounding q^to- 
plasm prevents the diffusion of undigested substances, such as proteins 
and starches, until they have been broken down into diffusable com- 
pounds of much smaller dimensions. 

The formation of food vacuoles by means of food cups (Kepner and 
Taliaferro, 1913; Schaeffer, 1916) is of interest in connection with a 
consideration of their surface structure. In Amoeba the membrane sur- 
rounding the food particles has been observed to be derived directly 
from the surface of the organism i.e., the plasmalemma. Schaeffer 
(1916) reports that A. proteus may form several hundred such food 
vacuoles a day, a fact which would necessitate considerable replacement 
of the plasmalemma. If this observation be correct, one need only as- 
sume a structure for the food vacuole membrane like that of the plasma- 
lemma. Furthermore, it has been shown that the membrane of a food 
vacuole which has completed its function in the cell may fuse upon eges- 
tion with the cell membrane from which it has been derived ( Rowland, 
1924c). In addition, some vital stains, such as neutral red, readily pene- 
trate the surface membranes of many Protozoa and stain the food vacu- 
oles, which suggests similar permeable properties for both the mem- 
branes surrounding the surface and those surrounding the food vacuoles. 
In contrast to its penetration of the surface and food-vacuole mem- 
branes, neutral red does not readily diffuse through the membranes 
of the nucleus or that of the contractile vacuole. 

Dissection and multilation studies upon a number of Protozoa have 
shown that the food vacuoles are capable of existing free of the cyto- 
plasm for relatively long intervals of time. For instance. King and 
Beams (1937) have observed food vacuoles in water to retain their 
form for over one-half hour, after which time the vacuolar membrane 
was observed to wrinkle, followed by a breakdown of the vacuole. 

Dogiel and Issakowa-Keo (1927) immersed Paramenum in various 
salt solutions and India ink. In solutions of MgSO^, MgCL, and FeSOj 
the food vacuoles are much elongated. These sausage-like food vacuoles 
may swell up or may be extruded through the gullet. In BaClg the food 
vacuoles are small and spindle-shaped. Whether this effect is produced on 
the membrane of the food vacuole or upon the cytoplasm is not clear. 

Mast (1938b) has recorded for A. proteus that a food vacuole may 



PROTOPLASM OF PROTOZOA 71 

divide several times, forming a number of vacuoles. In other cases food 
vacuoles have been observed to fuse (Mast and Hahnert, 1935). 

From the comparatively few accounts of the physical structure of the 
food vacuole membrane, it is not possible to give an analysis of its struc- 
ture. However, it seems reasonable to conclude that it is usually of mo- 
lecular dimensions, capable of resisting deformation, and that it is 
permeable to enzymes, water, certain dyes, and digested food materials. 
Its architecture is probably much the same as that of the surface mem- 
brane, except perhaps for its thickness. 

5. Other types of vacuoles. — In addition to the contractile vacuolar 
system and the food vacuoles, many other types of vacuoles may be found 
in the cytoplasm of the Protozoa. Such vacuoles may be characteristic 
of the cytoplasm, as the crystal vacuoles in Amoeba (Mast and Doyle, 
1935a), transitory vacuoles (Hopkins, 1938), large acid-filled vacuoles 
associated with changes in specific gravity of Noctiluca (E. B. Harvey, 
1917; Lund and Logan, 1925); or they may be induced by certain ex- 
perimental methods, such as change in the salt content of the environ- 
ment (Schaeifer, 1926), exposure to dyestuffs. X-rays, poisons, and so 
forth (Heilbrunn, 1928). These vacuoles, too, frequently have been 
reported to fuse; normally as in the coalescence of vacuoles in Noctiluca 
(Lund and Logan, 1925), and experimentally in Paramecium in which 
King and Beams (1937) observed the crystal vacuoles to fuse when cen- 
trifuged. 

Little is known concerning the actual physical structure of the mem- 
branes of such vacuoles, but there is no reason for believing that their 
structure differs greatly from that of other surface membranes surround- 
ing vacuoles, such, for instance, as the feeding vesicles of the contractile 
vacuole of a form like Euplotes or Varamecium. 

ADHESIVENESS OR STICKINESS 

Among the physicochemical properties of the protoplasm-liquid 
medium interface, adhesiveness is of importance in considering such 
subjects as ameboid movement, tissue culture, leucocyte activity, im- 
munity, and cell movements in embryology. Pfeiffer (1935) has listed 
the literature on adhesiveness, which is scattered very widely. 

It is well known that amoebae creep on vertical surfaces, even on the 



72 PROTOPLASM OF PROTOZOA 

under side of the surface film of water, creeping on this as though it 
were a sohd body, but Bles (1929) denies that Arcella can move on a 
clean surface film. Specimens of Amoeba on the under side of the cover 
glass swell up when disturbed, but may still stay attached to the "ceil- 
ing" (Bellinger, 1906). Adhesion to the substratum, according to 
Mast (1926b), is due either to the secretion of an adhesive substance or 
to a state of the plasmalemma. According to Rhumbler (1898) and 
Jennings (1904), an amoeba probably adheres to the substratum by a 
mucus-like secretion. Many observers have reported that if the surface 
of an amoeba is touched with a fine glass rod, it adheres to the glass 
rod, so that sometimes a bit of the organism may be pulled off (Mast, 
1926b) or the whole organism may be dragged about by a thread of 
mucus from the amoeba which has become attached to the glass rod 
(Rhumbler, 1898; Jennings, 1904). According to Chambers (1924), a 
stationary amoeba accumulates a considerable amount of slime, by 
which it is attached to the substratum; an amoeba dragged out of posi- 
tion and then released will be pulled back toward its original position. 
However, Schaeffer (1920) has not been able to convince himself that 
amoebae secrete mucus; nevertheless, whatever the method, the tips of the 
pseudopods often adhere so firmly to the substratum that strong squirts 
from a pipette are necessary to dislodge them (Bellinger, 1906). Hy- 
man (1917) applied a needle to the posterior end of an amoeba and 
pulled the animal in two, in spite of the tensile strength of the ecto- 
plasm. 

It is commonly held that attachment is one of the important factors 
in ameboid movement: when the organism is not attached there is no 
locomotion, although protoplasmic flow and the gel-sol and the sol-gel 
processes may be observed. Mast (1929) has studied the factors in- 
volved in attachment of A?7ioeba to the substratum and finds that simple 
agitation of the dish in which the amoeba are cultured may cause an in- 
crease in the firmness of adhesion. There was slight attachment in pure 
water (see also Parsons, 1926), but strong attachment over a great 
range of hydrogen-ion concentration, p\l 4.6 to 7.8). All the salts 
tested by Mast caused a decrease in time for attachment, and an increase 
in firmness of attachment; since non-electrolytes have no eff^ect, Mast 
believes that charges on the ions may change the surface charge of the 
amoeba or may change unknown internal forces. Bles (1929) has shown 



PROTOPLASM OF PROTOZOA 73 

that under conditions of low oxygen tension, Arcella releases its attach- 
ment and floats to the surface. 

According to Chalkley (1935), locomotor activity and attachment to 
the substratum are important factors in cytoplasmic fission in Amoeba. 
If one of the daughter cells is detached while the other remains attached, 
fission is not completed, but the unattached daughter flows back into the 
attached. However, if both amoebae are detached, as in distilled water, 
fission may be completed by the pushing of the pseudopodia of one ani- 
mal against those of the other, thus breaking the narrow cytoplasmic 
bridge between the daughter cells. 

In Testacea with lobose pseudopods, such as Dif^ugia and Centro- 
pyxis, the organism is pulled along by means of contraction of pseudo- 
pods, the tips of which have become attached to the substratum (Bellin- 
ger, 1906). That this attachment is of special nature and of considerable 
strength has been shown by Mast ( 1931a) , who prevented the shell from 
being dragged along by the contraction of the pseudopods, under which 
conditions the pseudopods were torn loose and snapped back toward the 
shell. Testacea, such as Difjiugia, Lesquereusia and Pontigulasia, attach to 
Spirogyra and devour the cell contents (Stump, 1935). According to 
Penard (1902), the filose pseudopods of such forms as Pseudodifjiugia 
and Cyphoderia have as their principle function the fastening of the ani- 
mal to the substratum, to which they adhere with extraordinary tenacity. 
Acanthocystis ludibunda (Helizoa) moves by adhesion of its axopods 
to the substratum, after which they contract, rolling the animal along 
over a distance twenty times its diameter in one minute (Penard, 1904) . 
Schaeffer (1920) has estimated that the axopods of this form must ad- 
here, contract so as to pull the animal along, and relax their hold, all in 
the short time of two seconds. The reticulose pseudopods of the Forami- 
niferan, Astrorhiza Umicola, attach and contract much as do the lobo- 
podia of the Testacea. Here, however, the organism leaves a trail of 
slime and bits of pseudopodia behind it, according to Schultz (1915). 
In Grom'ia squamosa the reticulose pseudopods play a very slight part 
in locomotion, but serve mainly as organs of attachment and food cap- 
ture (Penard, 1902). 

The whole outer layer of many rhizopods is sticky: it is well known 
that certain shelled rhizopods collect foreign particles to be included in 
their tests. According to Stump (1936), Pontigulasia will not reproduce 



74 PROTOPLASM OF PROTOZOA 

unless shell materials are present in the cultures in which the organisms 
are grown; shell materials are collected just previous to division, but 
before nuclear changes have begun. Verworn (1888) has found that 
only after mechanical irritation of the pseudopodia of Dijjlugia do they 
become sticky enough for glass particles to adhere to them, to be later 
drawn into the shell. Foreign particles may adhere to the surface of a 
moving amoeba and be carried forward by the outer layer, sometimes 
making many complete revolutions. Parsons (1926) has observed that 
carmine granules adhere to and move over the surface of an amoeba 
floating in distilled water, although there has been a definite loss of ca- 
pacity for adherence to substratum. Then, too, the outer layer of Fo- 
raminifera and Heliozoa is quite fluid, serving to capture food organisms 
which adhere to the surface. Certain of these organisms (i.e., Acti- 
nophrys) have been reported to form temporary colonies and to capture 
large objects of prey; such temporary colonies may be induced by me- 
chanical means (Looper, 1928). However, it has been shown by Daw- 
son and Belkin (1929) and Marsland (1933) that the adhesion between 
A. dubia and an oil surface is distinct from the process of ingestion, 
since a cap of oil may adhere to and spread over the tip of a pseudopod 
without ingestion taking place. The relation of adhesion to phagocytosis 
has been extensively studied in the amebocytes of the invertebrate Meta- 
zoa (see Faure-Fremiet, 1930; Loeb, 1927, 1928). 

A remarkable example of the stickiness of protoplasm is shown by 
the Choanoflagellata, in which the flagellum is surrounded by a proto- 
plasmic collar; food particles, brought to the collar by the movements 
of the flagellum, adhere and are carried to the point of ingestion by the 
flowing protoplasm of the collar. Some flagellates [Heteromita and 
Oikoinonas) have pseudopodia which are primarily adhesive in func- 
tion. While flagella are primarily organs of locomotion, they frequently 
are used as organs of attachment. This is commonly observed in Costia, 
Chilomonas, Heteromita, Pleuromonas, Anisonema, and Vetalomonas. 
The nature of this adhesion is not known. 

Certain of the developmental forms of trypanosomes become at- 
tached by their flagella to the walls of the organ of the invertebrate host 
in which they are found. Lwoff (1934) has mentioned that Strigomonas 
oncopelti and 5". jasciculata may swim free in cultures or may fasten to 
the glass side of the culture dish; they release upon a few seconds heating 



PROTOPLASM OF PROTOZOA 75 

at 55° C. On the other hand, in Leptomonas ctenocephali the attached 
forms are held in position by the sHmy enlarged tip of the flagellum, 
which is much shortened; they are killed at 55° C. before they can re- 
lease their hold. 

Adhesion phenomena have recently been introduced for diagnosing 
trypanosomiasis. In adhesion tests a drop of Trypanosoma suspension 
is added to one drop of equal parts of blood and 2-percent sodium cit- 
rate. If the blood comes from an infected animal, the red blood cells, 
and occasionally blood plates as well as bacteria, adhere to the trypano- 
somes. The immune serum contains an antibody that apparently acts 
upon the surface of trypanosomes to make it sticky. This test has been 
recently used by Taliaferro and Taliaferro (1934) in connection with 
other tests in equine trypanosomiasis; the adhesion phenomenon may 
persist for more than two years in animals that have been infected, 

Faure-Fremiet (1910) has emphasized the fact that while motility 
is the more general, it is not the only property of cilia. They may be- 
come immobile and serve as rigid stalks, or may be reduced to short rods 
and serve for fixation or for protection to the cell that bears them. It has 
frequently been observed that the movements of ciliates often cease tem- 
porarily when they come in contact with a firm surface. According to 
Jennings (1906) "the cilia that come in contact with the solid cease 
moving, and become stiff and set, seeming to hold the Paramecium 
against the object." Saunders (1925), however, holds that partially 
extruded trichocysts with sticky tips serve as temporary attachments in 
Paramecium. The hypotrichs generally can creep along vertical surfaces 
and on the under surfaces of the cover glass. The hypotrich Ancystro- 
podium maupasi may attach itself by its posterior cirri (Faure-Fremiet, 
1908) , and the holotrich Hemispeira by a bundle of fixative cilia (Faure- 
Fremiet, 1905). Kahl (1935) thinks that such adhesion depends upon 
the ability of cilia to become sticky, at least at the tips. Cilia are associated 
with attaching organs in Trichodina and in Ellobiophrya (Chatton and 
Lwoff, 1923a). Chatton and Lwoff (1923b) have also described organs 
of attachment derived from the posterior ciliated region in the Thigmo- 
tricha, Faure-Fremiet (1932) has studied the fixatory apparatus of 
Strombidium calkinsi which is constituted by two dorsal membranelles 
nearly as long as the body and made up of coalesced cilia which separate 
from each other at the adhesive distal extremity. At times the ciliate 



76 PROTOPLASM OF PROTOZOA 

seems to be walking on the solid surface, the two membranelles moving 
one after the other, somewhat like the cirri of the hypotrichs. The mem- 
branelles are not contractile but very elastic; the Stromhidium may un- 
fasten itself all at once and swim hastily away. Similar fixing organs have 
been described for other oligotrichs (5", urceolare and S. clavellinae) . 
Metacystis lagenula retracts by means of a large filament (a modified 
cilium glazed with sticky protoplasm), which adheres to the interior of 
the test, while M. recurva and Vasicola gracilis seem to be kept in their 
tests by a specialized cilium, a veritable bristle, which adheres to the 
inside of the test (Penard, 1922). Mesodin'nim pulex may adhere by 
means of tentacle-like structures, and various species of Stentor attach 
themselves to the substrate by means of cilia (Faure-Fremiet, 1910) 
which form together with ectoplasmic extensions the so-called pseudo- 
podia of the attaching disk of this form. The presence of slime-like or 
mucus secretions for attachment have been described for Spirostomum 
(Jennings, 1906), Urocentrum turbo and for Strombilidium gyrans. In 
the latter the slime, which is secreted by an attaching organ, the scopula, 
derived from modified cilia, congeals upon contact with the water into 
a very resistant thread which is attached to some structure in the medium 
(Faure-Fremiet, 1910; Penard, 1922). The Strombilidfum may then 
swing back and forth like a pendulum, held in position by the thread 
of slime which can be seen only because small particles of debris in the 
culture medium adhere to it. Certain observations of Chambers and 
Dawson (1925) on Blepharisma seem to show that the ability of cilia 
to combine into composite organelles, such as undulating membranes, 
membranelles, cirri, and so forth, may be dependent upon the presence 
of a slime-like secretion which spreads over the cilia and joins them into 
what looks like a homogeneous structure. At any rate these composite or- 
ganelles are often seen to break down into their constituent cilia, after 
which they may be recombined. This separation of composite motor 
organelles into their components often takes place upon fixation. 

According to Mast ( 1 909 ) , the prey of Didin'mm is held by means of 
the seizing organ, which in some way adheres to the surface when con- 
tact is made. Capture and ingestion of food depend upon the adherence 
of the seizing organ of Didinium and the strength of the ectoplasm of 
its prey, which is usually Paramecium. There are two different explana- 
tions of how the tentacles of the Suctoria adhere to their prey: by the 



PROTOPLASM OF PROTOZOA 77 

presence of a viscid secretion, or by active suction of the hollow tentacles. 
Hov^ever, the character of the outer surface of the prey is an important 
factor in its capture (Root, 1914). 

Specific Gravity or Density 

Whole organisms. — Since their specific gravity is slightly greater than 
that of water, most unicellular organisms can remain suspended in fresh 
or salt water only by the use of special locomotor organs such as cilia 
or flagella. Among the advantages of this slightly greater specific gravity 
is that the organisms are not caught in the surface film, nor in the con- 
gealing water on the surface when their locomotor activities are depressed 
because of low temperature; then, too, the simple methods of locomotion 
of Protozoa would hardly suffice to move bodies of very great density or 
to keep them suspended against the pull of gravity. In this connection the 
work of Jensen (1893) should be mentioned. In spite of the inaccuracy 
of his absolute measurements, it is clear that a 'Paramecium can lift 9 
times its own weight in water. Probably a Paramecium with a specific 
gravity above 1.35 could not keep itself in suspension because a very in- 
significant amount of energy 1 1 of the total is available for 

* 100 ~ 1000 

locomotion in this form (Ludwig, 1928a, 1930). 

Certain Protozoa of floating habit frequently have hydrostatic devices 
which aid in flotation, such as the gas bubbles secreted in the protoplasm 
of Arcella and Difflugia; special layers of vacuolated protoplasm, such 
as the calymma of Radiolaria; or very highly vacuolated protoplasm, as 
in Noctiluca and in Heliozoa. 

According to Bles (1929) the gas bubbles of Arcella are formed in 
the marginal protoplasm and are filled with oxygen. They are secreted 
when the oxygen tension is reduced experimentally and are adaptive, in 
that they reduce the specific gravity of the organism so that when oxy- 
gen tension is low the organism may float to the surface, where the oxy- 
gen tension is always somewhat higher. When Arcella is turned upside 
down so that the external pseudopods cannot adhere to the substratum, 
gas bubbles appear in from three to six minutes, before the animal be- 
gins to right itself and aid in this process by lowering the specific gravity. 
The bubbles disappear rapidly after the righting process is completed. 
When there is more than one bubble present all grow at the same time 



78 PROTOPLASM OF PROTOZOA 

and decrease at the same time. In the Radiolaria periodic migrations take 
place to and from the surface layers of the sea; these are brought about 
by changes in the vacuolar contents of the hydrostatic layer, which, ac- 
cording to Brandt (1885), is lighter than water. Schewiakoff (1927) 
has described the presence of a clearly defined gelatinous hydrostatic 
layer in the Acantharia (Radiolaria). Although the specific gravity of 
Noctiluca (1.014) is less than that of sea water (1.026) in which it 
floats, according to Massart (1893), E. B. Harvey (1917) has shown 
that this form can lessen and increase its specific gravity in a regulatory 
fashion. Lund and Logan (1925) have shown that the increase in specific 
gravity, following strong mechanical shock or electrical stimulus, is 
caused by the coalescence of large vacuoles and the liberation of their 
contents, which diffuse through the pellicle. The density of the solution 
in the vacuoles is less than that of sea water; according to Ludwig 
(1928b) this is because of its lower salt content, its osmotic pressure 
being about half that of sea water. Most marine animals possess body 
fluids almost isotonic with the external fluid, but Noctiluca, together with 
the marine teleosts, are hypotonic and, to a great extent, osmotically 
independent. Such organisms must be impermeable to water, absorb 
water in some way without salts, or take in sea water and excrete salts. 
Marine teleosts apparently take in sea water and excrete the excessive 
salts, and, since the membrane of Noctiluca is permeable to water, os- 
motic work (negative osmotic force) must be done by the membrane 
(E. B. Harvey, 1917) in maintaining this steady state instead of os- 
motic equilibrium. 

The protoplasm of marine Protozoa frequently becomes much vacu- 
olated upon transfer to fresh water. The marine variety of Actinophrys 
sol, according to Gruber (1889), has thick, granular protoplasm poor 
in vacuoles and entirely lacking a contractile vacuole; during gradual 
transfer to fresh water the protoplasm becomes foamy with bubbles and 
a contractile vacuole appears, so that the organism is indistinguishable 
from the fresh-water variety. The formation of vacuoles and the entrance 
of water into them undoubtedly lowers the specific gravity in this form, 
when transferred from salt to fresh water. The spine-like pseudopodia 
of Heliozoa, Radiolaria, and other floating forms also serve as a protec- 
tion against sinking. 

The first estimation of the specific gravity of a protozoan apparently 



PROTOPLASM OF PROTOZOA 79 

was that of Jensen (1893), who attempted to determine the energy rela- 
tions of the movement of P. aurelia. He obtained the value of 1.25 by 
suspending the organisms in solutions of potassium carbonate, a proce- 
dure which gave too high values because of excessive shrinkage, due to 
osmotic pressure. Later Piatt (1899) suspended killed or anaesthetized 
Paramecium and Spirostomum in solutions of gum arable and found 
their specific gravity to be 1.017. Lyon (1905) centrifuged living Para- 
mec'ium in solutions of gum arable and obtained 1.048 or 1.049. This 
was repeated by Kanda (1914, 1918), who finally arrived at a value of 
1.0382 to 1.0393 for Paramecium and 1.028 for Spirostomum. Fetter 
(1926) utilized approximately the same value, 1.038, which she ob- 
tained by centrifuging Paramecium in sugar solutions, in calculating 
the protoplasmic viscosity of that form. 

Leontjew (1927) has determined the density of various Protozoa 
{Fuligo, Stemonitis, slime molds; NaegJeria, an amoeba; and Dunaliella, 
a flagellate) to be 1.020 to 1.065. Some of his readings on Fuligo varians, 
obtained with a micropyknometer, are interesting enough to be men- 
tioned in detail: in moist weather the density was 1.01 6; in dry, 1.040, 
and 11 hours before spore formation, 1.065. 

Heilbrunn (1929a, 1929b) used 1.03 as the specific gravity of the 
protoplasm in his studies on viscosity of A. dubia. Motile amoebae 
{Naegleria) have a density of 1.043, according to Leontjew (1926a), 
and cysts I.O6O to 1.070 (Joschida, 1920, cited by Leontjew, 1927); 
cysts of Hartmanella hyalina, a soil amoeba, have a specific gravity of 
1.084 (Allison, 1924). 

It is, of course, generally recognized that the protoplasm of encysted 
Protozoa contains less water than that of active forms. Allison (1924) 
determined the specific gravity of cysts of Colpoda by the time required 
to fall through water. He finds that four-day cysts averaging 40.1 microns 
in diameter, have a density of 1.042; while twenty-day cysts, averaging 
25.1 microns, have a density of I.06I. Similar results were found for 
cysts of Gonostomum. The decrease in size and increase in specific grav- 
ity are apparently caused by water loss. 

The specific gravity of protoplasm other than that of Protozoa has 
been found to vary from about 1.02 to 1.08, with average values about 
1.045. The publication of Pfeiffer (1934) gives a resume of the meth- 
ods and results of such studies. 



80 PROTOPLASM OF PROTOZOA 

Relative specifc gravity of cell inclusions and components. — It has 
long been known that the various inclusions of protozoan cells are of 
different specific gravities. In centrifuging cultures to obtain large num- 
bers of organisms for fixation previous to morphological studies, it is 
often noticed that certain crystals have been displaced centrifugally. Mc- 
Clendon (1909) was one of the earliest workers to fix and stain Parame- 
cium after long-continued centrifuging; he found that the crystals and 
nucleus were displaced centrifugally. Heilbrunn (1928) mentions that 
a centrifuged Euglena loses its spindle-shaped contour and becomes 
spherical, with the granular inclusions packed at the centrifugal end. 
The same author (Heilbrunn, 1929b) has used the speed of movements 
of crystals centrifugally through the cytoplasm of A. dubia to estimate 
the absolute viscosity of the protoplasm; the specific gravity of the 
crystals was estimated to be approximately 1.10. E. N. Harvey (1931) 
records that the crystals of A. dubia fall down so rapidly that their 
velocity can hardly be determined in the microscope-centrifuge; and 
that the crystals of Paramecium were rapidly thrown down, as was the 
nucleus. He was also able to cleave living Stentor into two parts in the 
microscope-centrifuge; the lighter, oral half contained none of the 
Zoochlorellae which had been moved into the basal part. E. N. Harvey 
and Marsland (1932) observed the movement of cytoplasmic particles 
through the protoplasm of A. dubia and found them to be layered out in 
the following order: coarse granules and crystals, most centrifugal; nu- 
cleus, a visibly empty zone, a zone of fine granules, and, most centripetal, 
the contractile vacuole. Mast and Doyle (1935b) have recorded as 
follows the relative specific gravities of the various cytoplasmic com- 
ponents in A. proteus, from centrifugal to centripetal: refractive bodies, 
beta granules (mitochondria) and food vacuoles containing little or no 
fat, nucleus and food vacuoles containing much fat, hyaline protoplasm, 
contractile vacuole, crystal vacuoles without crystals, and fat globules. 
The position of the crystal vacuoles varies with the size of the included 
crystals: those with large crystals are heavy and move centrifugally in 
the centrifuge; those with small crystals are lighter. The small alpha 
granules, which are about 0.25 micron in diameter, are not layered out. 
All the refractive bodies, a large proportion of the crystals, and all the 
fat may be removed, with no injurious effects, from a centrifuged 
amoeba by cutting off the light and heavy ends. However, removal of 



PROTOPLASM OF PROTOZOA 81 

the beta granules (mitochondria) resulted in the death of the amoeba. 
Singh (1939) has also centrifuged A. proteus 'Y,' and found the order of 
layering to be: nutritive spheres, nucleus, crystals, neutral red bodies, 
mitochondria, cytoplasm, contractile vacuole, and fat. 

Patten and Beams (1936) centrifuged Euglena and found that the 
chloroplasts form a middle belt, having on the centrifugal side paramy- 
lum and neutral red bodies, while the clear cytoplasm containing the small 
spherical mitochondria is at the centripetal pole. In Menoidium the 
heaviest inclusions are the paramylum and neutral red bodies; in Chi- 
lomonas the starch and neutral red bodies are heaviest. Johnson (1939) 
has confirmed the results of Patten and Beams; in Euglena rubra, how- 
ever, hematochrome is present and is displaced to the centripetal pole 
with mitochondria. 

King and Beams (1937) ultracentrifuged Paramecium; in this form 
the various components and inclusions were layered in the following 
order from centrifugal to centripetal: crystals in vacuoles, compact 
chromatin of the macronucleus, food vacuoles and neutral red inclusions, 
achromatic matrix of the macronucleus, endoplasm, large clear vacuoles, 
and fat. Here the chromatin may be removed from the achromatic matrix 
of the macronucleus; the chromatin regenerates a macronucleus; the 
achromatic matrix persists for some time and apparently interferes with 
subsequent divisions. Browne (1938) has ultracentrifuged Spirostomum 
and has found the contents of the cell to be layered as follows : centrif u- 
gally located are the mitochondria, food vacuoles, and macronucleus; cyto- 
plasm; Golgi bodies; and centripetally, vacuoles. 

Daniels (1938) has used the ultracentrifuge in a study of gregarines; 
here the paraglycogen and chromidial granules are heaviest; next, the 
mitochondria and nucleus; cytoplasm; then the larger Golgi bodies; and 
lightest the smaller Golgi bodies and fat globules. In the gregarines 
studied the karyosome moved centrifugally in the nucleus, and the con- 
tents of the deutomerite layered independently of those in the primlte 
because of the presence of the transverse septum. 

It is obvious that the centrifuge may serve as an important research 
tool for the identification and study of the form, relative volume, and 
other characteristics of the components and inclusions found in proto- 
plasm. For example, Holter and Kopac (1937), by cutting amoebae in 
half after centrifuging, were able to demonstrate that the enzyme dipepti- 



82 PROTOPLASM OF PROTOZOA 

dase is apparently associated with the cytoplasmic matrix, independent of 
all cytoplasmic constituents which could be stratified by centrifugal ac- 
celeration. 

Optical Properties 

The ordinary optical characteristics of protoplasm, such as its trans- 
parency, color, and refractive index, do not seem to be of very great im- 
portance except that the observation of living protoplasm is conditioned 
by these properties. Too many have assumed that because a structure 
cannot be seen in living protoplasm, it is therefore nonexistent. 

TRANSPARENCY 

The ■ protoplasm of the protozoan cell is generally transparent or 
translucent, but in the presence of granular or other inclusions it may 
appear to be opaque or nearly so. Many of the differentiations are so 
nearly of the same index of refraction that special fixing and staining 
methods are necessary in order to study them. The state of aggregation 
of the colloids of the general protoplasm seems to be dependent, at least 
to some extent, on the salt content of the surrounding medium. Thus 
Actinophrys sol in sea water is densely granular, while in fresh water it is 
alveolar and translucent (Gruber, 1889). Spek (1921) has shown that 
Actinosphaerium becomes relatively opaque in certain salt solutions, and 
that P. bursaria, which is glass-clear, becomes dark brown in artificial salt 
solutions, owing to the collection of albuminoid substances into large 
aggregates. Certain observations of Schaeffer (1926) on marine Amoebae 
are of interest here: Flabellula pellucida, a most transparent marine 
amoeba, becomes densely granular in 2 5 -percent sea water, while F. 
cHata, another marine amoeba, is unusually transparent in 364-percent sea 
water. 

COLOR 

Protoplasm is usually observed to be colorless or grayish; many of the 
shades of blue, green, or yellow described for Amoebae are merely dif- 
fraction phenomena or subjective in nature. The color of the endoplasm 
of various amoebae has been described by Schaeffer (1926) as pale 
bluish-green, yellowish-green, bluish-gray; Hyalodiscus elegans has endo- 
plasm which is cirange-yellow centrally and ashen-gray peripherally. The 



PROTOPLASM OF PROTOZOA 83 

contents of the contractile vacuole are often slightly pink. This may be 
regarded as an optical illusion, since the color observed is complementary 
to the usual bluish-green of the endoplasm. Very frequently the color 
is obscured by the presence of colored inclusions of various kinds, or 
caused by colored inclusions of the same index of refraction as the proto- 
plasm and therefore difficult to differentiate. 

Ciliates may be colorless, gray, pink, blue, or violet. The blue color 
of 5". coeruleus is caused by a coloring matter, called stentorin, diffused 
through the cytoplasm, but in Blepharhma the color, which may vary 
from none through pink and violet to purple, and varies with the cul- 
tural conditions and from individual to individual in the same culture, 
\s apparently concentrated in the pellicle (Nadler, 1929). According 
to Jennings (1906), most colorless Infusoria do not react at all to a 
light of ordinary intensity; this has not been tested with forms such as 
Blepharhma, in which the color varies. 

In the plant-like flagellates color is usually caused by chromatophores, 
which may be green, blue (Lackey, 1936), brown, or yellow. The most 
interesting colored inclusion is the hematochrome, found in such forms 
as E. rubra as red granules from 0.3 to 0.5 microns in diameter. These 
euglenae form a green scum in shaded places; the green chloroplasts 
mask the hematochrome, which is centrally located; in direct sunlight the 
scum is red, the hematochrome being peripherally located and masking 
the chloroplasts. Control of the distribution of the hematochrome is so 
delicately balanced that if the euglenae are shaded for fifteen minutes, 
they change from red to green (Johnson, 1939). The mechanics of 
this control needs to be investigated. 

REFRACTIVE INDEX 

Even the finest strands of protoplasm can be seen in water, in spite 
of the fact that they may be transparent and colorless. This is because 
of their relatively high index of refraction. It is surprising that so little 
is known about the optical characters of protoplasm which may be seen 
to change during cell division. Schaeffer (1926) has shown that the 
nuclei of certain marine amoebae become much more prominent by dilu- 
tion of the sea water with fresh water, and Chalkley (1935) has shown 
that there is a change in the refractive index of the nuclei of A. pro tens 
during division: the interkinetic nuclei can easily be seen at a magnifica- 



84 PROTOPLASM OF PROTOZOA 

tion of 200 diameters; those in division cannot. Very few measurements 
of the refractive index of protoplasm have been made; Frederikse 
(1933b) has reported a value of 1.40 to 1.45 for A. verrucosa; Mackin- 
non and Vies (1908) 1.51 for the cilia of Stentor, and 1.56 for the 
flagellum of Trypanosoma [Sphochaeta) halhiani. Mackinnon and Vies 
made their determinations by immersing the organisms in media of 
different refractive indices; double refraction, due to depolarization, 
disappears in media of the same refractive index as the cilia and flagella. 
Faure-Fremiet (1929) found the index of refraction for entire amoebo- 
cytes of Lumhricus to be 1.400, for the hyaloplasm 1.364; of Asterias 
to be 1.446 and 1.385 respectively (for methods see Pfeiffer, 1931). 

Structural Properties 

It has long been known that the polarity of a cell may persist after 
the relative positions of its various visible constituents have been changed; 
this has led to the idea that polarity has its basis somehow or other in 
the structure of homogenous cytoplasm, which remains unchanged in 
spite of exposure to high centrifugal forces (Conklin, 1924), Polarity 
and symmetry are generally present in the Mastigophora and Infusoria, 
in which an anterior-posterior axis is usually persistent throughout active 
life, and has been described as present in cysts (Lund, 1917, in Bursar/a) . 
However, in Sarcodina such as Amoeba, polarity may be thought of as 
continually changing, being bound up with the gel-sol process at the 
temporary posterior end, the flow of protoplasm forward, and the sol- 
gel process at the temporary anterior end. Hyman (1917) has demon- 
strated that the temporarily differentiated anterior end of Amoeba is the 
region of highest susceptibility to cyanide. Mast (1931) and others 
have reported that electrical currents have a solating action on the 
plasmagel, on the side directed toward the cathode. 

Recently Chalkley (1935) has studied the process of cytokinesis in 
A. proteus. He observed, with the onset of prophase, a loss in sensitivity, 
a swelling up of the organism, a decrease in activity of the contractile 
vacuole, and an increase in movement of the granules in the region 
of the nucleus. With the separation of the daughter chromosome plates, 
he observed a flow of the cytoplasm from the equator, in the same direc- 
tion as the separating daughter plates. As the daughter plates approach 
the surface of the cell and the new nuclei begin to form, a solation of 



PROTOPLASM OF PROTOZOA 85 

the plasmagel takes place, resulting in the formation of numerous 
pseudopodia which become attached and undergo active ameboid move- 
ment. At the same time, because of the flow of the cytoplasm from the 
equator toward the two poles, the region of the Amoeba at the equator 
has become narrowed to a thin neck. Presumably a solation of the 
plasmagel at the equator, together with a pull exerted by the two ac- 
tively dividing ameboid daughter cells, produces the final separation of 
the organisms. The temporary polarity becomes immediately lost after the 
completion of division. 

The physical-chemical factors involving the change in polarity of the 
protoplasm at the equator and the forces responsible for the flow of the 
protoplasm from this point are unknown. However, Chalkley thinks 
the fundamental principles involved in these processes are the same as 
those described by Mast for ameboid movement, namely sol-gel trans- 
formation. Further, it has been shown by Chambers (1938) that if the 
nucleus is moved toward the cell surface, pseudopodia are induced in 
that region. Becker (1928) has demonstrated that the factor which de- 
termines the direction of streaming and hence polarity is located in 
Mastigina, a flagellated amoeba, in the region of the nucleus. If the 
nucleus is moved posteriorly, streaming ceases and is then resumed to- 
ward the nucleus. 

It should be pointed out that in the Protozoa nuclear division and 
cytoplasmic fission may be closely correlated or widely separated in tempo, 
and that they often exhibit a considerable degree of independence. How- 
ever, the plane of separation of the nucleus usually determines the plane 
of cytokinesis, in that they usually take place at or nearly at right angles 
to each other. In organisms like the Mastigophora, the plane of nu- 
clear division is parallel to the anterior-posterior axis and coincides 
with the plane of cytokinesis of the organism. In Ciliata the plane of 
nuclear division is perpendicular to the anterior-posterior axis and coin- 
cides with the plane of cytokinesis. 

In some ciliates there appears to be a definite and permanent divi- 
sion zone laid down early in the life of the organism, which is not 
disturbed by diverse multilations of the body (Calkins, 1926). Further- 
more, in Fronton/a this zone differs so markedly from the surrounding 
cytoplasm that it can be easily seen in the living condition (Popoff, 
1908). 



86 PROTOPLASM OF PROTOZOA 

Multilation studies by Calkins (1911) and by Peebles (1912) on 
Paramecium have resulted in the production of numerous monsters, 
Peebles describes this condition as due to an upset in nuclear and cyto- 
plasmic division tempo; thus when the nucleus is ready to divide, the 
cytoplasm is not, and vice versa. If this be true, the mitotic and cyto- 
kinetic phenomena in this form must be closely integrated, and the 
division mechanism of the organism as a whole be dependent upon the 
proper coordination of both the nuclear and the cytoplasmic division 
processes. 

Child and Deviney (1926) and Child (1934), have shown that in 
ciliates generally there is an anteroposterior gradient, due to the existence 
of a physiological gradient in the longitudinal axis. The anterior end is 
more susceptible to many agents, and there is also an axial differential 
in the rate of reduction of methylene blue. Child is of the opinion that 
this metabolic gradient is the only basis of physiological polarity. Lund 
(1917, 1921) has found that reversal of polarity often occurs in cut 
halves of Bursar ia undergoing regeneration; it may also occur in normal 
animals. An indication of this change in polarity was a reversed beat of 
the cilia. He further found that Paramecium showed a reversed beat of 
cilia, in direct electrical currents of proper strength. Verworn (1899) 
has shown that paramecia and other ciliates orient themselves with the 
anterior end of their bodies toward the cathode to which they swim. 
On the other hand, many flagellates show an opposite behavior. 

Schaeffer (1931) has presented evidence that the protoplasm of the 
amoebae, and presumably of other organisms, consists primarily of spe- 
cific molecules which are organized into definite patterns, and that most 
or all of the characteristics of the organisms are due to or correlated 
with positional relationships of the molecules. 

It is generally thought at the present time that adjacent protein mole- 
cules, because of their multipolar character, have an orienting efi^ect 
upon one another and that the resulting configuration may be equivalent 
to a net-like structure, extended in three dimensions. That this is true 
may be inferred from the anomalous viscosity of solutions of proteins 
and protoplasm: they show non-Newtonian flow, i.e., their viscosity 
varies with the stress applied, although they may outwardly conform to 
true fluids in being free from rigidity. 

Bensley (1938) has recently isolated from the cytoplasm of liver 



PROTOPLASM OF PROTOZOA 87 

cells a material called plasmosin, which he thinks is constituted of linear 
micelles. 

The formation of a fiber results from the end-to-end orientation of these 
linear micelles. In protoplasm in the liquid state these micelles are probably 
independent and irregularly arranged but in flowing protoplasm they would 
be oriented parallel to the axis of flow. . . . From this state by simple 
end-to-end combination all changes in viscosity are possible up to the forma- 
tion of a fibrous gel . . . or even discrete fibers. 

Bresslau (1928) has also shown that "tektin," a material extruded by 
ciliates, has anisotropic properties somewhat similar to those of plasmo- 
sin. 

The fact that the chromosomes probably represent gene-strings has 
been of enormous importance in determining our ideas of significant pro- 
toplasmic structure. The chromosomes apparently reproduce themselves 
at each cell division, so that their individuality is retained in all cell 
generations. The demonstration of the presence of these linear aggregates 
of visible size, which are self-perpetuating, cleared the way for the micel- 
lar theory of protoplasm structure. 

Much of the evidence for the presence of linear aggregates in proto- 
plasm has been obtained by microdissection, by the use of dark-field and 
polarization microscopes, by studies on cohesion and swelling, and by 
X-ray dififraction methods. Some of these have already been considered 
and others will be discussed below. 

ELASTICITY 

According to Seifriz (1936), "elasticity is the best indication we have 
of the structure of living matter" and is evidence for the presence of 
linear aggregates. A body is said to be elastic if after having been strained 
it tends to return to its original form when the stress is removed. Volume 
elasticity is characteristic of fluids and solids; shape elasticity (rigidity) 
of solids and colloids in the gel condition generally. The form assumed 
by the bodies of various flagellates and ciliates is characteristic and 
offers means of identification in many instances. Relative rigidity is of 
common occurrence among those Protozoa, such as Euplotes, which have 
a dififerentiated pellicle and a firm ectoplasm. C. V. Taylor (1920) 
demonstrated this elasticity by applying pressure with a microneedle; the 
body bent conspicuously over the needle but returned to normal shape 



88 PROTOPLASM OF PROTOZOA 

upon release of the pressure. Animals cut two-thirds across may keep 
their shape; this argues for a stiff consistency of the ectoplasm, as well 
as a tough pellicle. Any apparent modification in the shape of E. patella 
occurs only from outside pressure, since the animal is unable to vary its 
shape. Other hypotrichs may be similarly armored, as is shown by the 
fact that they are broken in the ultracentrifuge (King and Beams, un- 
published work) so that fragments are found swimming around as 
though the whole animal were brittle rather than plastic. In other forms, 
such as Paramecium, the body may be constricted when the animal 
forces its way through obstacles. Upon ultracentrifuging in gum solutions 
paramecia become much elongated and thin, because of the presence of 
materials of different specific gravities in the cell. Such elongated animals 
may survive and return to their normal shape unless the pellicle and 
ectoplasm have been strained beyond the limit of their elasticity, in 
which case the structure responsible for return to normal shape has been 
destroyed and they die, permanently deformed (King and Beams, 1937) . 
That the form of Paramecium is determined by the relatively firm outer 
layers has been shown by some observations of Chambers (1924), who 
tore the ectoplasm with a microneedle. The fluid interior pours out into 
the surrounding water and the ectoplasm soon disintegrates; but oc- 
casionally the fluid endoplasm forms a delicate surface film which main- 
tains the integrity of the extruded mass. Merton (1928) has studied 
these so-called autoplasmic paramecia which, deprived of pellicle, cilia, 
trichocysts, and ectoplasm, take on the form of a fan-shaped amoeba, 
which may live for some days, divide, and exhibit locomotor activities. 
Under unfavorable conditions a rayed stage, with long pseudopodia-like 
extensions reminiscent of a. A. radiosa, may be assumed. That the pellicle 
is not the only element involved in rigidity has been shown for Blephar- 
hma by Nadler (1929). The pellicle of this form may be shed after im- 
mersion in weak solutions of strychnine sulphate. The "naked" animal 
emerges from the old pellicle with the shape and elasticity characteristic 
of the species. Eventually the pellicle is reformed, and the process may 
then be repeated. 

That amoebae have elasticity of form to a considerable degree has 
been shown by Jennings (1904), who bent a pseudopod with a glass 
rod; when released the pseudopod sprang back into its original position. 
Whole amoebae were also bent, with subsequent return to original form 



PROTOPLASM OF PROTOZOA 89 

after release; Dellinger (1906) and Hyman (1917) have repeated these 
experiments, as indeed may be done by anyone. Rowland (1924c) has 
stretched the outer layer of A. verrucosa with microneedles; upon re- 
lease the animal recovers its normal shape, apparently unharmed. The 
protoplasm of plasmodia of slime molds (Seifriz, 1928) is at times 
poorly elastic, and at other times it may be stretched into very fine, long 
threads which snap back a goodly distance when released. 

Seifriz has also determined elastic values by inserting minute nickel 
particles into the protoplasm of slime molds and attracting these particles 
electromagnetically. On release of the current the metal particles return 
to their original position; the distance traveled is measured and used as 
an indicator of elasticity. A maximum stretching value of 4.4 microns 
was obtained for liquid, previously streaming protoplasm of myxomy- 
cetes, a maximum value of 292 microns for quiescent, highly viscous 
exuded masses of protoplasm from plasmodia. This latter value is slightly 
greater than that for gelatin solutions and slightly greater than that of 
fresh egg albumen. 

The long thread-like pseudopodia of Foraminifera, which usually pull 
the organism along by adhering to the surface terminally and then con- 
tracting, have been shown to be elastic by Schultz (1915), who cut 
these and observed them to snap back like a rubber band. 

The reticulose pseudopodia (myxopodia) of the Foraminifera are very 
different from the lobose and filose forms in other Rhizopoda. The 
former have a soft miscible outer protoplasm which leads to fusion on 
contact with one another and a relatively rigid inner axial structure 
which shortens without wrinkling when the pseudopod is withdrawn. 
As this denser core is formed as an elongation "in the direction of 
growth, strains will be set up during the process which will give rise 
to ordered and preferential arrangement tending toward the crystalline 
state" (Ewles and Speakman, 1930). Thus it will be seen that the axial 
solid protoplasm of these myxopodia, although it is not in the form of 
a fiber, serves the same function as the axial filament of the Heliozoa 
and Radiolaria. 

Up to this point elasticity has been considered principally in connec- 
tion with the protoplasm itself, or in its temporary completely reversible 
structures. There remain for consideration those differentiations which 
last the whole life of the organism and are usually irreversible, such as 



90 PROTOPLASM OF PROTOZOA 

flagella, axial filaments, cilia, myonemes, and supporting fibers (mor- 
phonemes) of various kinds. The fact that these are elastic is too well, 
known to need more than mention here. Spindle fibers, on the other 
hand, are often thought to be artifacts, but Cleveland (1935), work- 
ing on the hypermastigote flagellates, has pulled the centriole out of 
position; the chromosomes were also displaced, but both centriole and 
chromosomes immediately sprang back into position when released. This 
argues not only for the reality of the chromosomal and spindle fibers, but 
also that they are structures of considerable elasticity. If one considers 
the aphorism of Needham (1936) "that biology is largely the study of 
fibers," these fibrillar structures of the Protozoa are of great interest 
because they consist of parallel aggregations of the submicroscopic elon- 
gated particles (micelles) of protoplasm (see Taylor, infra, Chapter IV) . 

CONTRACTILITY 

We may distinguish between active contraction, as in muscle fibers, 
and elastic shortening after having been stretched, as in elastic fibers in 
the higher animals. Although there are many examples of contractility 
and elasticity, in the Protozoa, associated with differentiated myonemes or 
morphonemes respectively, there are also many instances of active con- 
traction in the absence of any optically differentiated structure. Accord- 
ing to Lewis (1926), theories of contractility must be based on the 
presence of a contractile molecule, because the fibrillae seen in heart 
muscle in tissue culture are not "true" cytological structures but are 
due to reversible gelation. Faure-Fremiet (1930) also holds that the 
gelified condition is often bound up with the existence of internal 
fibrillar structures, which disappear when solution occurs. That such 
fibrillae appear and disappear may, of course, be caused by aggregation 
and disaggregation of smaller invisible fibrillae, or may even be due to 
changes of refractive index. It is well known that objects that are of the 
same refractive index, transparency, and color cannot be seen, even in 
the dark field (Schmidt, 1929). 

It is generally assumed that contraction of the gelled ectoplasmic 
cylinder in Atnoeba forces the more fluid endoplasm forward (Schaeffer, 
1920; Pantin, 1923; Mast, 1926b). The contraction is thought to be 
caused by the fact that the gel-sol process at the posterior end of the 



PROTOPLASM OF PROTOZOA 91 

amoeba results in an increase of volume and so stretches the gelled cyHn- 
der of protoplasm; the resulting elasticity forces the sol forward where a 
decrease in volume has occurred upon gelation of the plasmasol. How- 
ever, in shelled forms, such as Difflug/a (Dellinger, 1906; Mast, 1931a) 
and Centropyxis, there is an active longitudinal contraction of the plas- 
magel cylinder which results in locomotion by pulling the shell along. 
That the circular elastic contraction in Amoeba and the longitudinal 
active contraction in Dijjiug'm are different mechanisms may be doubted. 
However, Mast (1931a) has shown that Dijjiugia deprived of their 
shells move much as does Amoeba. It is to be noted that the source of 
energy is in the ectoplasm, so that the streaming of the endoplasm in 
these forms must be of a different nature from cyclosis in other forms 
such as Paramecium or Frontonia, where protoplasmic streaming is ex- 
tremely difficult to explain in terms of contraction. 

There are many examples of local contractility in the literature of 
ameboid movement. Swinging and revolving movements of lobose 
pseudopods when not in contact with the substrate have been described 
by Penard (1902), Jennings (1906), Hyman (1917), Kepner and 
Edwards (1917), and many others. These differential local contractions 
of the ectoplasm often approximate muscular activity, according to Kep- 
ner and Edwards. Schaeffer (1926) has described corkscrew-shaped pseu- 
dopodia in Astramoeba jlageWpoda, with from two to eight spirals which 
wave about quite like flagella, often making a complete revolution in 
three seconds. 

The most spectacular instances of local contraction are those in which 
an Amoeba pinches a large ciliate in half. Mast and Root (1916) de- 
scribe this process as taking ten seconds for Paramecium and show that 
it cannot be explained in terms of the surface tension of the Amoeba. 
Beers (1924) describes the constriction of Frontonia by Amoeba until 
the former was dumbbell-shaped, and ascribes the pinching to centripetal 
pressure exercised by an extending collar of protoplasm which pinched 
the prey in half in eight minutes. Kepner and Whitlock (1921) saw 
a partly ingested Paramecium constricted much as described by Beers for 
Frontonia, except for the loss of the cilia from the ingested part. The 
figures and descriptions of the two latter instances are very similar to 
those of Grosse-AUermann (1909) for ingestion by invagination in 



92 PROTOPLASM OF PROTOZOA 

A. terrkola, and of Mast and Doyle (1934) for the ingestion of water 
by various species of Amoeba in albumin solutions. Penard (1902) has 
described and figured the pinching off of an extensive injured portion 
by A. terricola as has Jennings (1906) for A. Umax. The process of 
egestion, as figured by Howland (1924c) for Amoeba verrucosa, seems 
also to involve extensive local contraction. 

The lobopodia and filopodia of the Amoebida and Testacea are solid 
peripherally with a central fluid region, while the pseudopods of the 
Radiolaria and Heliozoa (axopodia) and those of the Foraminifera 
(myxopodia) are more fluid peripherally and more solid axially. In 
axopods and myxopods there can be no flow of endoplasm caused by 
the elastic contraction of a gelled ectoplasmic cylinder. Roskin (1925) 
describes the origin of the axopods of Actinosphaerimn by the flowing 
together and alignment of fibrillae, which eventually fuse into a hollow 
tube, filled with fluid which is associated with rigidity and contractility. 
That these axopods do contract rapidly has been shown for Acanthocystis, 
which, according to Penard (1904), may traverse twenty times its own 
diameter by rolling along the tips of the axopods, which must adhere, 
contract, and then release very rapidly. In the foraminiferan Astrorhiza, 
according to Schultz (1915), pseudopods stretch out five to six times 
the length of the body, make "feeling" movements and may finally either 
adhere to the substratum or contract and be withdrawn. The organism is 
usually fastened by three bundles of pseudopods; if one of these be torn 
loose, the others contract rapidly and the animal is pulled forward. The 
axial, more solid stereoplasm of these pseudopodia is distinctly fibrillar. 
Schmidt ( 1929) has also described the formation of contractile pseudopo- 
dia in the Foraminiferan Khumbler'inella by the alignment and coales- 
cence of fibrillae. 

The minute size of cilia and flagella makes it extremely difficult to 
determine whether their movements are due to active contraction or to 
changes occurring in the cell which bears them. 

In addition to their usual method of locomotion by means of flagella, 
many Mastigophora such as the euglenoids show euglenoid movement, or 
metaboly. In these forms there are present in the pellicle more or less 
spiral striations, which apparently are elastic in nature and tend to pre- 
serve the form of the organism during and after the contraction of the 
superficial layers of the body. In some cryptomonads (e.g., Chroomonas 



PROTOPLASM OF PROTOZOA ,93 

pulex) springing movements are brought about by the strong contrac- 
tion of the outer layer of the body, the resulting locomotion being inde- 
pendent of flagellar movement. Certain flagellates have an extraordinary 
superficial resemblance to medusae both in appearance and in method of 
locomotion: (Cystoflagellata: Leptodiscus, Craspedotella; Phytomona- 
dida: Medusochloris; Dinoflagellata: Clipeodin'ium). In these types ac- 
cording to Pascher (1917), the movements represent a special form 
of metaboly and two mechanical systems should be present, one radial 
and dilating, the other peripheral and contracting. The former could 
of course be elastic only and bring about passive return after the con- 
traction of the latter. 

In the Infusoria there are many examples of specialized retractile 
organelles: the tentacles of Suctoria may be retracted and extended much 
as may the axopods of the Heliozoa. The structure of these tentacles is 
quite similar to that of the axopods ( Roskin, 1925). The remarkable 
tentacles of the ciliate Actinobolina may be extended to a length twice 
the diameter of the body or may be completely retracted. They are as- 
sociated internally with two groups of fibrils which seem to wind up to 
retract and unwind to extend them (Wenrich, 1929). In many ciliates, 
such as Stetjtor and Spirostotiium, there are actively contractile myonemes 
in the ectoplasmic layer. In other related forms, such as Climacostomum, 
the corresponding structures are elastic only and have been referred to 
as morphonemes. The myonemes may even appear to be striated (Dierks, 
1926, and others) but this has been denied (Roskin, 1923). 

A most unusual case of extension and retraction occurs in the ciliate 
Lacry^nana olor. Here the "neck" may be extended to fifteen times the 
length of the body, the form of which remains unchanged. This "elas- 
ticity" is associated with the presence of what appears to be a series of 
spiral striations. However, upon complete extension of the neck, Penard 
(1922) has observed that there is only a single continuous spiral. No 
experimental work has been done upon this form, so that the nature of 
the extension and retraction is not understood. However, Verworn 
(1899) has cut the neck of L. olor free from the head and body. The 
neck retains its extensile and contractile properties exactly as when in 
connection with the body. 

The finer structure of the contractile stalks of the Vorticellidae has 
been studied by Koltzoff (1912), Fortner (1926), and many others. 



94 PROTOPLASM OF PROTOZOA 

The stalk consists of an external wall, an inner liquid, and a spirally 
wound contractile cord. Within the contractile cord is an excentrically 
placed myoneme often called a spasmoneme, the contraction of which 
causes the stalk to become coiled like a spring. The distal end of the 
stalk is usually attached to the substratum, but in Vorticella natans and 
V. mayen the organisms are never attached but swim, stalk first, through 
the water. In the former the stalk rolls up into narrow spirals, but in the 
latter the stalk swings in a wide loop on contraction somewhat like a 
flagellum. Belehradek and Paspa (1928) have reconstructed a myogram 
from moving pictures of the stalk of a vorticellid and find that the 
spasmoneme does not function like a true muscle but like a modified 
flagellum. Various attempts have been made to explain the contraction 
and extension of the stalk of vorticellids as caused by the complex action 
of two opposing bundles of fibers, or in terms of internal pressure against 
coiling brought about by elastic fibers. 

Myonemes are also well developed in the gregarines, where longi- 
tudinal and circular myonemes are apparently responsible for bending 
and peristalsis-like movements. Myonemes called myophrisks are also 
present in certain Radiolaria. Here they are associated with the spreading 
out of the gelatinous cortical layer, previous to their decrease in specific 
gravity and subsequent rise to the surface (Schewiakofi^, 1927). 

ROPINESS, OR THREAD FORMATION 

Living material is said to be ropy if it can be drawn out into threads; 
ropiness is thought to be due to the micellar structure of the material. 
The ability of a drop of a pure liquid to resist distortion is due to its 
surface tension only, and in heterogenous mixtures the surface film may 
approximate a solid consistency. A column of fluid breaks up into a 
number of smaller spherical bodies. The formation of pseudopodia, 
especially of filopodia, axopodia, and myxopodia, demonstrates the pres- 
ence of solid structures in protoplasm. Then, too, protoplasm may be 
drawn out into fine strands of considerable elasticity and tensile strength: 
the ectoplasm of Amoeba has been drawn out into fine strands by Hyman 
(1917) and many others; Schultz (1915) was able to draw out the 
protoplasm of the foraminiferan Astrorhiza into long threads; this was 
more marked in the outer layers than in the inner mass of protoplasm. 



PROTOPLASM OF PROTOZOA 95 

Many other observers, using microdissection methods, have confirmed 
this for a whole series of animal and plant cells. The spinning of threads 
from protoplasm is generally assumed to be dependent upon the sub- 
microscopic fibrillar structure of the protoplasm, the latter being respon- 
sible for its elasticity and high degree of extensibility. The tensile strength 
of a strand of myxomycete protoplasm has been found by Pfeffer (quoted 
by Seifriz, 1936) to be 50 mgm. per square millimeter. The ropiness of 
protoplasm is of course conditioned by temperature, viscosity, hydrogen- 
ion concentration, and other factors which affect protoplasm (review 
byjochims, 1930). 

Further evidence of the micellar nature of protoplasm may be deduced 
from the experiments of Seifriz (1936), who has examined the proto- 
plasm of slime molds with a Spierer lens. When the protoplasm is quiet 
it presents a mosaic appearance, but when in active flow or when formed 
into a thread is presents a striated appearance. Harvey and Marsland 
(1932) noted that the crystals of Amoeba fall in jerks when moving 
under centrifugal force; this may be due to the presence of structural 
elements in the protoplasm. Moore (1935) has found that slime molds 
in the plasmodial stage will flow through pores 1 p in diameter. He also 
forced this living material through bolting cloth of various mesh sizes. 
The Plasmodia survived after being forced through pores 0.20 mm. in 
diameter or larger, but died when forced through smaller pores. Moore 
thinks that there are fibrillar elements in slime mold protoplasm which 
are destroyed if forced unoriented through a pore through which they 
could flow if the micelles were properly oriented. 

DOUBLE REFRACTION 

The great advantage of the use of the polarizing microscope is that 
characteristic structure which is otherwise imperceptible may be revealed, 
even in the living organism, without any alteration of the specimen. 
Amorphous and pseudoamorphous materials (i.e., materials in which 
the orientation of the particles is a random one) are dark when viewed 
between crossed nicol prisms. True double refraction is always the re- 
sult of an orientation of optically anisotropic elements; many substances, 
such as glass, become doubly refractive if deformed by external forces. 
Many sols when in movement or when placed in an electric or a magnetic 



96 PROTOPLASM OF PROTOZOA 

field show double refraction, as do many gels when under pressure or 
when drawn out into threads. Mechanical stress may produce double 
refraction in gelatin even of 0.01 percent. 

Phenomena of double refraction in protoplasm often indicate the 
presence of mechanically and optically anisotropic elements oriented in 
some definite way, and are among the best evidences for the presence 
of micelles in protoplasm. See Schmidt, 1937, for a complete discussion. 

Valentin saw double refraction in the cilia of Opdina ranariim in 1861 
and Rouget in the stalk muscle of Cctrchesium in 1862. Engelmann 
(1875) carried out very extensive pioneering work on double refraction 
on the Protozoa and other forms. He saw anisotropy in the pellicle 
of large ciliates, such as Opdina, but was unable to distinguish double 
refraction in the myonemes of Stentor because the entire outer layer is 
doubly refractive. Mackinnon (1909) mentioned that the protoplasm of 
Actinosphder'mm is quite generally anisotropic, and Schmidt (1937) 
saw faint but unmistakable traces of double refraction at the edge of the 
surface layers of Amoeba, which became distinctly greater as the amoeba 
became rounded up before encysting, the cyst wall showing it very 
distinctly. 

Engelmann (1875) also observed that the axopods of /I r//«cj-/'/?<im»;w 
were doubly refractive in the living condition, the anisotropy being 
coextensive with the axial filaments which extend deep into the proto- 
plasm. With the withdrawal of the pseudopods the condition vanishes. 
Mackinnon (1909) confirmed the findings of Engelmann. The structure 
of the axial filaments of these pseudopods is known to be fibrillar (Ros- 
kin, 1925; Rumjantzew and Suntzowa, 1925). Schultz (1915) found 
the fibrillae in the rhizopods of the foraminiferan, Astrorhha, to be 
doubly refractive, and Schmidt (1929) found weak anisotropy in the 
axopods of the radiolarian, Thalasskolla. Schultz (1915) and Schmidt 
(1929) report that the stereoplasmic axis of the pseudopods of the 
Foraminifera are formed by the parallel alignment of fibers. 

Cilia have been found to be doubly refractive by Valentin, Engel- 
mann (1875), and Mackinnon and Vies (1908). The latter authors 
consider this to be caused by the depolarization of the light by reflection 
from surfaces of the cilia, because of the difference of refractive index. 
If air penetrates into the axis of a cilium, upon drying the anisotropy is 
greatly increased (Schmidt, 1937). The so-called rootlets of the cilia 



PROTOPLASM OF PROTOZOA- 97 

are doubly refractive, but the basal granules are not (Engelmann, 
1880.) Myonemes have been quite generally shown to be anisotropic, 
especially those in the stalk muscle of the vorticellids (Engelmann, 1875; 
Wrzesniowski, 1877; Mackinnon and Vies, 1908). The myoneme of 
the stalk spreads out into fibrillae in the base of the animal. These, too, 
are doubly refractive. Engelmann (1875) found that the extensile neck 
of Trachelocerca [Lacrymaria) olor, when stretched out, was positively 
anisotropic in relation to its longitudinal axis. It is to be recalled that 
the neck may be extended as much as fifteen times the length of the 
body. Associated with this is a single spiral thread which becomes 
straight upon extension of the neck (Penard, 1922). 

Brandt ( 1885 ) has shown that the isospore nuclei of the Radiolaria are 
anisotropic, but not the vegetative nor the anisospore nuclei. This has 
been confirmed by Schmidt (1932) on living and preserved material 
of the same form. Schmidt (1929) had also observed double refraction 
of the nuclear membrane in living nuclei of a foraminiferan and of 
Amoeba; in the latter he also observed weak anisotropy of small visible 
granules (chromatin?) in the living nucleus. Finally, Kalmus (1931) 
has recorded that certain elements of the division figure of the nuclei 
of Paramecium show slight traces of double refraction during fission and 
conjugation. 

X-RAY DIFFRACTION AND ULTRACENTRIFUGATION 

Early studies on cohesion and swelling relations of organic fibrillar 
structures indicated that the finer structures of which they are composed 
are micellar in nature. Recently X-ray diffraction methods have sub- 
stantiated this view and have made it possible actually to measure the 
dimensions of these structural units. Much of this work has been done 
on keratin, elastin, chitin, myosin, cellulose, and other nonliving sub- 
stances. Some observations have been made on living nerve fibers 
(Schmitt, Bear, and Clark, 1935). 

The evidence from X-ray diffraction shows that animal fibers owe 
their anisotropic properties to the fact that they are composed of 
longitudinally oriented protein chains. However, in most protoplasm the 
configuration of such chains must be such that it may be altered rapidly 
and reversibly. For a review of X-ray diffraction, see Frey-Wyssling, 
1938. 



98 PROTOPLASM OF PROTOZOA 

Beams and King (1937) and King and Beams (1938) have shown 
that the comphcated process of karyokinesis may occur in eggs of 
Ascaris, although cytokinesis does not usually take place at very high 
centrifugal forces. They believe that if stratification occurred, it would 
involve a breakdown of the normal, submicroscopic spatial relations, 
which are of importance for the maintenance of life. 

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Taylor, C. V. 1920. Demonstration of the function of the neuromotor ap- 
paratus in Euplotes by the method of microdissection. Univ. Cal. Publ. 
Zool., 19: 403-70. 

1923. The contractile vacuole in Euplotes: an example of the sol-gel 

reversibility of cytoplasm. J. exp. Zool., 37: 259-89. 

1935. Protoplasmic reorganisation and animal life cycles. Biol. Rev., 

10: 111-22. 

Taylor, H. S., W. W. Swingle, H. Eyring, and A. A. Frost. 1933. The effect 

of water containing the isotope of hydrogen upon fresh water organisms. 

J. cell. comp. Physiol., 4: 1-8. 
Tchakhotine, S. 1937. Radiations, cell permeability and colloidal changes. 

Trans. Faraday Soc, 33: 1068-72. 
Thornton, F. E. 1932. The viscosity of the plasmagel of Amoeba proteus at 

different temperatures. Physiol. Zool., 5: 246-53. 

1935. The action of Sodium, Potassium, Calcium, and Magnesium ions 

on the plasmagel of Amoeba proteus at difl^erent temperatures. Physiol. 
Zool., 8: 246-54. 

Tiegs, O. W. 1928. Surface tension and the theory of protoplasmic move- 
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Verworn, M. 1888. Biologische Protisten-Studien. Z. wiss. Zool., A6: 455-70. 

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Voegtlin, C, and H. W. Chalkley. 1935. The chemistry of cell division. IV. 
The influence of HgS, HCN, COg and some other chemicals on mitosis 
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Wrzesniowski, A. 1877. Beitrage zur naturgeschichte der Infusorien. Z. wiss. 
Zool., 29: 267-323. 



CHAPTER III 

CYTOPLASMIC INCLUSIONS 
Ronald F. MacLennan 

All active cells possess a large number of q^toplasmic granules 
which change in number, size, shape, and composition in accordance 
with the changes in the activities of the cell of which they are a part/ 

The fact that small granules are so constantly present in the living substance 
is an indication that such a fine suspension of material represents a colloidal 
condition favorable for the life process. It seems certain that as the physiology 
of the cell becomes more clearly understood there will be shown to be a 
definite dependence of vital phenomena on the granular nature of protoplasm, 
on the properties which it possesses by virtue of the fact that it is a suspen- 
sion (Heilbrunn, 1928, p. 20). 

The cytoplasmic granules are a visible part of the fundamental organi- 
zation of the cell, and the elucidation of their functions contributes not 
merely to a specialized branch of cytology but contributes directly to a 
solution of the fundamental problem of protoplasmic organization. 

The richness of the granular complex early attracted the interest of 
cytologists, and many studies were made on their chemical composition. 
The report of Biitschli's discovery that the carbohydrate granules of 
gregarines differ from those in vertebrates is one of the classic papers 
in the group. During the first thirty years of this century the emphasis 
shifted from the earlier cytochemical methods to an interest in certain 
of the granules as permanent, self-perpetuating cytoplasmic organelles, 
which could be classified by certain empirical reactions such as osmic 
reduction or the segregation of janus green and neutral red. Dissatisfac- 
tion with the specificity of these methods has resulted recently in a re- 
newed emphasis on methods which yield specific information on the 
chemical and physical nature of the cytoplasmic granules and their 
cyclic changes. Too often, however, there has been a tendency to carry 

' This paper is a contribution from the departments of Zoology of the State College of 
Washington and Oberlin College. 



112 CYTOPLASMIC INCLUSIONS 

on these two types of investigation separately, with the result that in 
many cases the morphological and functional studies of the cytoplasmic 
granules have become separated. The one group emphasizes the classifica- 
tion of granules into hard and fast categories of Golgi bodies, mitochon- 
dria, vacuome and so forth, with little specific consideration of function, 
while those engaged in functional studies tend to group all the granules 
together or to devise entirely new systems, which hinder the comparison 
of granules in several species. This review is an attempt to coordinate 
these two angles of approach, so that both may contribute to our under- 
standing of the role of the cytoplasmic granules in the cell. 

Since summaries are available of the characteristics of single groups 
of granules, this review is not intended to provide an exhaustive catalogue 
of the facts of any one group. Particular emphasis is placed on those 
granules which have been described with sufficient completeness to fur- 
nish evidence as to reactions, classification, and function, as well as their 
relationship to other granules in the same cell. Specific directions on 
standard techniques for demonstrating the various granules are avail- 
able in the various books on microtechnique and histochemistry and so 
are not described in detail here. The more recent publications will be 
emphasized, since the specificity of methods has improved greatly and 
summaries of the earlier papers are available in the works of Calkins, 
Doflein-Reichenow, and others. The Protophyta have been omitted in 
most cases, since their inclusion would complicate the picture unneces- 
sarily. 

Mitochondria 

Undoubtedly many granules described in early cytological studies of 
Protoza were actually mitochondria, but their status as a separate group 
of cytoplasmic constituents in the unicellular organisms was not recog- 
nized until the publication of the monograph of Faure-Fremiet (1910), 
which emphasized the concept that mitochondria are universal, self -per- 
petuating cytoplasmic constituents. 

The identification of mitochondria is not yet entirely satisfactory, 
since it depends upon stains and fixatives of the lipoid component, a 
material not restricted to mitochondria alone, or upon vital dyes which 
are not as efi^ective in the Protozoa as in the Metazoa and which in certain 
cases stain other organelles as well. Typical mitochondria are refractile 



CYTOPLASMIC INCLUSIONS 113 

in life, become grey brown, or black in osmic techniques, but usually 
bleach faster than the Golgi bodies, reduce pyrogallol, take basic stains 
after fixation in lipoid preservatives, stain weakly or not at ail after fixa- 
tives containing acetic acid, and are stained vitally with janus green B. 
This last method is often considered to be the final criterion, but un- 
fortunately in many cases the mitochondria of Protozoa stain only a pale 
green (Subramaniam and Ganapati, 1938, and others), not the dark 
green described in metazoan cells. In addition, the stain is not always 
specific, since Lynch (1930) found that any concentration from 1:2000 
to 1:500,000 tints the entire organism [Lechriopyla), although the 
mitochondria can be distinguished by their darker color. Hayes (1938) 
found that none of the granules in Dileptus stained electively with Janus 
green B. An additional source of difficulty is the fact that in flagellates, 
the parabasal bodies are often stained as darkly as the mitochondria. 
Although the parabasal bodies and mitochondria show additional simi- 
larities in staining reactions and composition, Volkonsky (1933) points 
out that the former are derivatives of the neuromotor system and cannot 
be considered as homologous with the mitochondria. 

Mitochondria present a wide variety of shapes, but most commonly 
they are spherules (Fig. 17), chains of spherules (Fig. 26), short rods 
(Figs. 22, 23), or dumb-bells. The filamentous structures found so often 
in metazoan cells are found rarely in the Protozoa, but a good example 
has been described in the phytoflagellate Polykrikos by Chatton and 
Grasse (1929). Lynch (1930), in his studies on the ciliate Lechriopyla, 
found a compound structure (Figs. 15. 16), composed of several discs. 
Each disc is composed of chromophobic material, with a rim of chromo- 
philic material which stains with Janus green and the other mitochondrial 
dyes. In cases of secretion (Fig. 17), the secretion granules often appear 
as a chromophobic center in the mitochondria (MacLennan, 1936), but 
the mitochondrial material itself usually appears to be homogeneous 
either in the living unstained ciliate or after any of the mitochondrial 
stains. It is probable that the chromophobic center of the discs of 
Lechriopyla represents material secreted by the rim, which is the mito- 
chondrial part of the complex discs. In some cases, however, mitochondria 
may have a true duplex structure, since Mast and Doyle (1935b) showed 
that the outer surface of the mitochondria of Amoeba stain more deeply 
than the center. This diff^erentiation may be explained either as a definite 



15 IG 



n 









24 



25 




21 ■?. 



-V* 


<^- 




•Jf 


22 


23 




26 



27 

Figure 15-16. From Lechriopyla mystax: Figure 1?, end view after Hirschler's 
mitochondrial technique; Figure 16, lateral view, after Champy-iron haematoxylin. (After 
Lynch, 1930.) Figure 17. From Ichthyophth'trius multlfilns, series showing mitochondria 
and the secretion of paraglycogen, vital stain with janus green. (After MacLennan, 
1936.) Figures 18-21. From Monocystii showing de novo origin of mitochondria: Fig- 
ure 18, sporozoite; Figure 19, trophozoite; Figure 20, conjugating gametes from a cyst; 
Figure 21, spores, Champy or Flemmings — iron haematoxylin. (After Horning, 1929- ) 
Figures 22-25. From Atnoeba proteus: Figures 22-23, free in cytoplasm; Figure 22, nor- 
mal: Figure 23, fixed in modified Regaud's fluid; Figures 24-25, on surface of contractile 
vacuole. (After Mast and Doyle, 1935.) Figures 26-27, From Aggregata ebe.rthi: Figure 
26, mitochondria proper; Figure 27, mitochondria associated with protein reserves. 
(After Joyet-Lavergne, 1926.) Figures 28-29. From Btirsaria truncatella: Figure 28, 
section of early conjugant; Figure 29, section of later conjugant. (After Poljansky, 
1934.) 

In all cases, material which responds to mitochondrial stains is drawn in solid black; 
associated granules are stippled. 



CYTOPLASMIC INCLUSIONS 115 

localization of stainable materials or as a dense surface with less dense 
centers, with the type of material the same in both places. 

The mitochondria in most Protozoa are fairly evenly distributed 
through the cytoplasm, sometimes alone and sometimes associated with 
various types of storage granules (see the discussion of function). 
Faure-Fremiet (1910) found that if any localization occurs, the mito- 
chondria tend to concentrate beneath the pellicle and occasionally around 
the contractile vacuole. Horning ( 1927) extended these observations and 
contends that mitochondria tend to concentrate near all membranes — 
particularly around the food vacuoles during active digestion, beneath 
the pellicle, and around the nucleus. Hall and Nigrelli (1930) criticized 
Horning's identification of mitochondria, which was based largely on 
dark-field observations, and showed that in Vorticella sp. the mito- 
chondria are not associated with the food vacuole. Volkonsky (1934) 
likewise rejected Horning's identification and showed that the granules 
associated with the digestive vacuoles, in a large number of species, are 
stainable only with neutral red. MacLennan (1936) found a similar 
situation in Ichthyophthirius. In other Protozoa, however, the accumula- 
tion of mitochondria near membranes has been confirmed. Mast and 
Doyle (1935b) find that the mitochondria in Amoeba are occasionally 
associated with the gastriole (food vacuole) and they were able to cor- 
relate the association with the type and stage of digestion. They also 
demonstrated an association with the contractile vacuole, confirming the 
earher work of Metcalf (1910). Volkonsky (1934) found a small ag- 
gregation of mitochondria around the food vacuole of Campanella and 
Paramecium during the alkaline phase of digestion. Chatton and Grasse 
(1929) showed that the filamentous mitochondria of Polykrikos tend to 
accumulate near the pellicle, but instead of being parallel to the surface, 
as in the ciliates described by Horning, are perpendicular to the surface. 
Poljansky (1934) studied the changes occurring in the life cycle of Bur- 
saria and found that the mitochondria of neutral individuals are uniformly 
dispersed throughout the cytoplasm, but during conjugation (Figs. 28, 
29) the mitochondria migrate to the periphery and form a definite zone 
under the ectoplasm, and also around the micronuclear derivatives. The 
chondriosomes again scatter during the growth of the macronuclear pri- 
mordium. There seems to be neither universal nor permanent localization 
of mitochondria near membranes, as is to be expected according to Horn- 



116 CYTOPLASMIC INCLUSIONS 

ing's theory that mitochondria accumulate at the intracellular surfaces in 
accordance with the Gibbs-Thompson Law. Doyle (1935) suggests that 
mitochondria tend to collect at regions of active interchange, since 
he found that mitochondria are the only granules which flow out into the 
pseudopodia of the foraminiferan Iridia. While this theory is attractive in 
certain cases, it is difficult to see how this would apply to the concentra- 
tions which occur in conjugating Burs aria. 

Mitochondria are more widely accepted as universal, permanent, and 
self-perpetuating granules than any other cytoplasmic component, and for 
this reason it is worth while to consider in detail the proof upon which 
such statements rest. Rigid proof of this theory requires a demonstration 
of mitochondria only, with, of course, a lack of evidence of any de novo 
origin. Furthermore, it is obvious that proof of the continuity of any 
cytoplasmic component cannot be based on a study of only one stage 
in the life cycle, but must rest upon adequate studies of the whole life 
cycle. 

Mitochondria have been identified in a multitude of Protozoa of all 
groups by Faure-Fremiet (1910) and later authors, and as a result these 
components are usually considered to be present in all Protozoa. How- 
ever, recent evidence shows that this assumption is unjustified. An ex- 
treme example is Trypanosoma diemyctyli, in which Nigrelli (1929) 
was not able to demonstrate any pre-formed mitochondria, although 
granules which satisfy the general criteria of mitochondria are induced 
by exposure of the organisms to Janus green B. These induced granules 
are not permanent, but disappear after about two hours. The marine 
amoeba Flabellula is another species in which mitochondria are normally 
lacking. Hopkins (1938b) found that the normal amoeba possessed no 
granules which can be classified as mitochondria, but that when the 
amoeba is disturbed in a variety of ways, granules are precipitated in 
small pre-formed cytoplasmic vacuoles, the contents of which are nor- 
mally a homogeneous fluid. After the recovery of the amoeba from the 
disturbing conditions, the granules are resorbed. These temporary 
granules possess the staining reactions of mitochondria, including the 
ability to segregate Janus green B, and are the only granules in this 
organism which can be classed in that group. As a contrast to these cases 
of induced mitochondria, Kirby (1936) reports the experimental destruc- 
tion of the mitochondria which are a normal component in the cytoplasm 



CYTOPLASMIC INCLUSIONS 117 

of flagellates from termites. In normal Pseudodevescovina, large num- 
bers of mitochondria can be demonstrated by the Flemming-Regaud 
method but, after feeding for three days on filter paper soaked in one- 
percent Janus green B, no mitochondria can be demonstrated. Addi- 
tional examples of the lack of mitochondria are furnished by studies of the 
whole life cycle of certain Protozoa. Horning (1929) was able to demon- 
strate mitochondria in most stages of Monocystis (Figs. 19, 20), but 
found that these granules disappear in the sporozoite stage (Fig. 21). 
Beers (1935) and MacLennan (1936) also found that mitochondria 
disappear in the later encysted stages of ciliates, although the same 
methods give positive results in other stages of the cycle. These observa- 
tions under both normal and experimental conditions demonstrate that 
mitochondria are neither universal nor permanent cytoplasmic constitu- 
ents. 

The crucial point in the classification of mitochondria as autonomous 
organelles is whether they always arise from preexisting mitochondria. 
The occurrence of dumb-bell-shaped mitochondria and other possible 
division stages have been found so often in fixed and stained preparations 
that it is unnecessary to quote this evidence here. The observations on 
living material are few and perhaps the clearest is that of Horning 
(1926), who reported division stages of mitochondria in a living 
heterotrich and was able to confirm the descriptions based on fixed 
material. Thus division is a factor in the increase in numbers of mito- 
chondria, but returning again to the studies of the whole life cycle, we 
find that division is not the only method, since mitochondria must be 
formed de novo (Figs. 18-21) in those species in which the mito- 
chondria have disappeared during the quiescent phases of the life 
cycle. Mitochondria are not self-perpetuating organelles, but are differ- 
entiations which may endure for a longer or a shorter period during 
the cycle of the cell. 

In the cases considered above, mitochondria are cytoplasmic in their 
origin, but this is by no means the only possibility. Joyet-Lavergne 
(1926) states that the group of mitochondria attached to protein granules 
(Fig. 27) are derived from the nucleus along with the protein reserves 
(for discussion of this point, see p. 163), but Daniels (1938) in the 
same or related species could find no mitochondria attached to the 
protein granules. Calkins (1930) found in Uroleptus one set of cyto- 



118 CYTOPLASMIC INCLUSIONS 

plasmic granules, which he traced back to macronuclear fragments dur- 
ing conjugation, interpreting these as mitochondria. Since the staining 
methods were not very specific and these granules do not stain with 
Janus green, the statement that these are mitochondria must be accepted 
with caution. Miller (1937) has endeavored to prove that mitochondria, 
Golgi bodies, and other cytoplasmic inclusions in A. proteus are "bac- 
teria spores, fungi, or yeasts, together with indigestible material of 
certain food organisms." This idea is, of course, similar to that of Wallin 
and has been so thoroughly criticized by Cowdry and others that it need 
not be considered here in detail. Miller was not able to culture these 
cytoplasmic "bacteria" and his main argument seems to be based on the 
observation that mitochondrial stains and Golgi type impregnations will 
demonstrate granules in the culture medium. This merely shows that 
the stains used are not always specific under all conditions, a fact which 
has been pointed out many times. Miller does not present any evidence 
which can stand up against the observations and experiments of Mast 
and Doyle (1935a, 1935b), Holter and Kopac (1937), and Holter and 
Doyle (1938) on the same species. The mitochondria are specializations 
of the cell itself, probably in all cases from the cytoplasm, and are neither 
artifacts nor invaders. 

The composition of mitochondria is still incompletely known in any 
exact sense, in spite of the large amount of work done on these com- 
ponents. They have long been thought to be composed of both lipoids^ 
and proteins, because of their staining reactions and solubilities (Faure- 
Fremiet, 1910; Hirschler, 1924, 1927). Unfortunately no one has 
yet repeated in Protozoa the work of Bensley (1937), who isolated 
mitochondria from liver and was able to make both qualitative and 
quantitative analyses. These analyses confirmed the cytochemical analysis 
of lipoid and protein, but instead of the large amounts of phosphatids 
and so forth, which were predicted from the cytological reactions, he 
showed that the lipoid is largely neutral fat. Bensley also found that 
many reactions (for example the osmic-acid reaction) are much weaker 

^ Lipoid is used here, as in most cytological works, in a very general sense. It includes 
all materials which are soluble in ether, absolute alcohol, and so forth, and which stain 
with Nile blue sulfate, the sudans, and other fat soluble dyes. Neutral fat, fatty acid, 
phosphatids, and the like respond to these tests. Lison (1936) suggests the rather 
awkward term sudanophil material, in order to emphasize the cytological side and to 
avoid false implications as to chemical nature. 



CYTOPLASMIC INCLUSIONS 119 

in mitochondria in the cytoplasm than in mitochondria isolated from the 
cytoplasm. These observations confirm the validity of the interpretation 
of mitochondrial reactions in the Protozoa as indicative of a lipoid- 
protein mixture, but emphasize the need of caution in more specific 
interpretations before methods as specific as Bensley's are applied to the 
Protozoa. It is clear that the evidence now available as to the nature of 
the lipoids and of the proteins in the mitochondria of Protozoa is sig- 
nificant largely as a lead for further work. 

Horning (1927) adheres to the view that the lipoid component of 
mitochondria is a phosphatid, but presents no conclusive evidence for 
this statement. Wermel (1925) found that the mitochondria (or lipo- 
somes) of Act'mosphaerium react with Ciaccio's method, but according 
to Lison the only valid interpretation is that unsaturated lipoids are 
present. MacLennan (1936) stated that the lipoid material in the mito- 
chondria of Ichthyophthirius is a fatty acid, on the basis of a blue stain 
with Nile blue sulphate, used according to Lorrain Smith's method. 
However, since Lison (1936) presents evidence against the specificity 
of this method, the above interpretation is perhaps too strict, but it is 
interesting to note that the original interpretation is in accord with 
Bensley's analysis of the mitochondria of liver. There is likewise a 
lack of information on the nature of the proteins present. Hayes (1938) 
demonstrated a positive reaction to fuchsin-sulfurous acid reagent and 
claimed that nucleic acid is present. However, since lipoids may react 
in this manner in the "plasmal reaction," it is possible that this test was 
concerned with the lipoid component rather than the protein portion. 

The metallic impregnation of mitochondria, or the depth of stain taken 
after the use of lipoid solvents, varies between the species of Protozoa 
and has usually been considered to be a rough indication of the pro- 
portion of lipoids present. Thus Scott and Horning (1932) find in 
Opalina a large amount of lipoid; Lynch (1930) in Lechriopyla, Patten 
(1932) in Nyctotherus, MacLennan and Murer (1934) in Paramecium, 
find some lipoid; while Beers (1935) in Didinium finds little if any 
evidence of lipoids in the mitochondria. Although this data is very 
crude, foundation is provided for the working hypothesis that there 
is a wide variation in the probable composition of mitochondria, rang- 
ing from practically pure lipoid to almost pure protein. Much of this 
difference represents constant differences between species and could be 



120 CYTOPLASMIC INCLUSIONS 

detected by Bensley's mass technique. However, Faure-Fremiet (1910) 
found staining differences within the mitochondria of a single indi- 
vidual, and Peshkowskaya (1928) reports that the ectoplasmic chondrio- 
somes of ClJmacostomum are resistent to fixatives which usually dis- 
solve mitochondria, although the endoplasmic mitochondria are much 
more typical in their reactions. Pellissier (1936) found similar dif- 
ferences, not within the cell but between various individuals, and was 
able to show that all the mitochondria impregnate more deeply in the 
vegetative stages than in the stages just before reproduction. By the 
selection of species in which all granules are in the same stage at the 
same time, Bensley's mass technique could be used very profitably 
in exploring the changes in mitochondrial composition. 

MacLennan and Murer (1934) found heavy deposits of ash in the 
typical mitochondrial rods, as well as in the other cytoplasmic granules 
of Paramecium. 

The presence of enzymes in mitochondria have been indicated in- 
directly in many cases by the morphological association of these bodies 
with structures in which digestive or synthetic activity is going on. 
The only direct demonstration of the localization of cytoplasmic 
enzymes is due to Holter and Kopac (1937) and Holter and Doyle 
(1938), who showed that dipeptidase is not present in mitochondria, 
but that amylase is. The method used was a combination of centrifugal 
localization of granules and micro-methods for the measurement of 
enzymatic activity. The mitochondria were concentrated in one end 
of an Amoeba, which was then cut and the enzymatic activity of the 
mitochondria-rich and the mitochondria-poor portions of the cytoplasm 
compared. Since both the centripetal and the centrifugal portions had 
the same amount of dipeptidase (measured by the ability to split 
alanylglycine) per unit volume of cytoplasm, Holter and Kopac con- 
cluded that the enzyme is in the matrix. They point out that this proves 
nothing as to the origin of the enzyme, which might diffuse out from 
a granule as fast as it is formed. Holter and Doyle found that the 
middle region of the centrifuged Amoeba had the most amylase 
(measured by the digestion of starch). The nucleus, crystals, digestive 
vacubles, mitochondria, and matrix are found in this zone of the centri- 
fuged Amoeba. The enzyme could not be localized in the nucleus, since 
non-nucleated fragments show no significant diminution in amylase, 



CYTOPLASMIC INCLUSIONS 121 

nor in the crystals since most of these are in the centrifugal end which 
would thus have the highest enzymatic activity. They also ruled out a 
localization in the digestive vacuoles by a demonstration that the enzyme 
content of hungry and feeding Amoeba, with a resultant difference in 
the number of vacuoles, is the same. The only structures the distribu- 
tion of which after centrifuging corresponds with the distribution of 
amylase are the mitochondria. The study of the centrifuged Amoeba 
presents many difficulties, since the stratification is never complete and 
there is always some mixing between the finishing of centrifuging and 
cutting the amoeba in parts, but the further use and development of these 
methods and their use in species in which stratification is complete 
will undoubtedly aid in the complete analysis of mitochondria and other 
cytoplasmic granules. 

The theory that mitochondria are concerned with cellular respiration 
has led to attempts to identify in the mitochondria the materials known 
to be active in this respect. One of these is glutathione, in which the 
physiologically active group is sulfhydril, demonstrable cytochemically 
by the sodium nitroprusside reaction. Joyet-Lavergne (1927-29) found 
that the mitochondria of Sporozoa give a positive reaction with sodium 
nitroprusside, and this was confirmed by Cowdry and Scott (1928) in 
Plasmodium. Chalkley (1937), however, found that the strongest re- 
action in vegetative Amoeba is in the nucleus and that at the metaphase 
this material is poured into the cytoplasm. Some granules in the nucleus 
gvwQ a particularly strong reaction, but in the cytoplasm the coloration 
is diffuse. These results of Chalkley's extensive work on glutathione in 
Amoeba suggest the desirability of a reinvestigation of the Sporozoa, 
and certainly indicate that the materials containing the sulfhydryl group 
are not always localized in the mitochondria. Joyet-Lavergne (1934) has 
also shown that the mitochondria give a strong reaction with the anti- 
mony trichloride test for vitamin A, and concludes that this is a part of 
the respiratory mechanism along with glutathione. Although respiration 
cannot be discussed in detail here, it should be pointed out that the 
glutathione-vitamin A theory presents many difficulties and the system 
more usually accepted is glutathione-ascorbic acid (Holmes, 1937). 
Bourne and Allen (1935) and Bourne (1936) have demonstrated the 
concentration of ascorbic acid in cytoplasmic granules by the acetic- 
silver-nitrate method, but unfortunately have not correlated these with 



122 CYTOPLASMIC INCLUSIONS 

any particular type of granules. Daniels (1938) used this method in 
gregarines, but although she was able to demonstrate granular accumula- 
tions in the cells of the gut, the gregarines remained clear. 

The functions ascribed to mitochondria in the Protozoa may be 
grouped under two main headings: synthesis (or segregation), and 
respiration. The first group includes a number of activities, all of which 
involve the accumulation of materials and in some cases the synthesis 
of new products from these raw materials. Examples of this type of 
function for which there is definite evidence are the secretion of reserve 
bodies, a digestive function in connection with the gastrioles, excretory 
function in connection with the contractile vacuoles, and the transport of 
materials from one organelle to another. Respiration also might be 
considered to fall in this category, since it would depend upon the ac- 
cumulation in the mitochondria of the substances responsible for the 
oxidation-reduction processes of the cell. 

The secretion of reserve bodies is cytologically the easiest phase of 
accumulation to demonstrate. Faure-Fremiet described the formation 
of deutoplasmic granules by direct transformation of mitochondria, as 
well as by the more common method of segregation adjacent to or 
within the mitochondria which retain their identity. Joyet-Lavergne 
(1926a) occasionally found a relationship of this type between the 
mitochondria and paraglycogen and always found that protein granules 
possessed a mitochondrial cap (Fig. 27), but denied that this indicated 
anything more than a casual relationship. Horning (1925) described 
mitochondrial rims around the protein granules in Opalina (this identi- 
fication of mitochondria is denied by Kedrowsky, 1931b) and accepts 
it as evidence of secretory activity. MacLennan (1936) described the 
origin of paraglycogen in the center of spherical mitochondria (Fig. 17) . 
The identification of these granules was made not only with the usual 
permanent stains for mitochondria, but with specific microchemical 
stains (Sudan III, Nile blue sulfate, iodine, chlor-zinc-iodide), and 
their growth observed in live specimens stained with Janus green. The 
fact that the paraglycogen first appears as a center in a solid mito- 
chondrial sphere refutes the usual suggestions that the secretion merely 
happens to be in contact with the mitochondria and that there is no 
real connection between the two. There is no evidence as yet to show 
whether these visible secretions are actually synthesized by the mito- 



CYTOPLASMIC INCLUSIONS 



123 



chondria or are the result of the segregation by the mitochondria of 
materials synthesized at other points. Wermel (1925) found that 
certain mitochondria of Actinosphaerium have a high lipoid content, 
as shown by Ciaccio's lipoid methods, i.e., a so-called liposome rather 
than an ordinary type of mitochondria, and concludes that they secrete 
the lipoid reserves. Except for the fact that these granules stain weakly 




Figure 30. The association of mitochondria with the gastriole in Amoeba proteus. 
(From Mast and Doyle, 1935b.) A, 2-6 hours; B, 6-8 hours; C, 8-16 hours; D and E, 
16-30 hours; b and bi, mitochondria; f, fat; s, starch; v, vacuole refractive bodies; c, 
crystals; p, pellicle of Chilomonas. 



with Janus green, they are like the intermediate lipoid bodies and could 
perhaps be classed more conveniently with them. Zinger (1928) 
counted the "spherical inclusions" and the mitochondria in Ophryoglena 
and found that they are roughly proportional in number. He concluded 
that the spherical inclusions are derived from mitochondria. Since many 
granules of entirely different origin will increase in number under 
favorable conditions, this conclusion cannot be considered as proved. 
The digestive function of mitochondria rests upon two types of 
evidence: the actual demonstration of enzymes in these granules (see 
p. 125), and the correlation between the periodic aggregation of mito- 
chondria around the gastriole and the type of digestion taking place 
within. Both types of evidence are available for A. proteus, so that the 
results of observations may be checked against a direct knowledge of the 



124 CYTOPLASMIC INCLUSIONS 

enzymes actually present. In A. proteus, Mast and Doyle (1935b) find 
that the mitochondria accumulate around the food vacuoles six to eight 
hours after its origin and then again at sixteen to thirty hours (Fig. 30) . 
During the first contact, digestion begins, starch is changed to erythro- 
dextrine, and fat leaves the vacuole. In the second contact, vacuole re- 
fractive bodies and crystals disappear. These authors never found the 
mitochondria actually entering the vacuole. The relationship between 
mitochondria and starch digestion vv'as directly demonstrated by Holter 
and Doyle, who showed that these granules contain amylase. Their 
function is restricted with respect to digestion, since they lack dipeptidase, 
according to similar studies by Holter and Kopac (1937). This situa- 
tion is not universal, since Hopkins (1938b) also working with Mast, 
showed that no formed granules are associated with digestion in the 
marine amoeba, Flabellula, although a material is dissolved in the 
vacuoles which when precipitated by disturbance, and so forth, forms 
granules which stain with Janus green B and other mitochondrial stains. 
Again, in many other Protozoa the mitochondria have no direct con- 
nection with digestion. Thus mitochondria may contain enzymes in some 
cases, but this is not a necessary association. 

Excretory granules associated with contractile vacuoles have been 
described many times, but only recently have mitochondria been proved 
to be associated with the excretory process. Mast and Doyle (1935a) 
have shown that the excretory granules of A. proteus (Figs. 24, 25, 47) 
correspond in their staining reactions to mitochondria. By centrifuging 
the majority of these bodies into one end of the amoeba and removing 
this part, these authors showed that the formation of the vacuole is 
dependent upon these granules and that if most of the mitochondria 
are removed, death follows. In one experiment, most of the mito- 
chondria were left, with the result that the average interval between 
pulsations was 3.46 minutes. If few mitochondria were left, the time 
was correspondingly longer, and when very few granules were left, 
the average time between pulsations increased to twenty-five minutes. 
Mast and Doyle interpreted these experiments as indicating that some 
excretory material, toxic to Amoeba, is eliminated by the vacuole, and 
that the mitochondria function as the means of transport to the vacuole. 

Doyle (1935) harmonized the apparently discrepant functions of 
digestion and excretion by pointing out that they may be united under 



CYTOPLASMIC INCLUSIONS 125 

the general heading of transport, and pointed out further that mito- 
chondria tend to accumulate wherever exchanges are taking place, both 
within the individual and at the outer surface. Mast and Doyle (1935b) 
observed that the mitochondria accumulate around the crystal vacuoles 
while the crystals decrease in size, and with the surface of the refrac- 
tive bodies while the latter are increasing in size. The mitochondria of 
Amoeba seem to be a mechanism for intracellular transport and for 
carrying amylase to the food vacuoles, digested material from the food 
vacuoles and crystal vacuoles to the refractive bodies, and metabolic 
wastes to the contractile vacuoles. However, since Holter and Kopac 
(1937) have shown that dipeptidase is not associated with mito- 
chondria, and Volkonsky (1933) and MacLennan (1936) have shown 
cases in which vacuome alone touches the food vacuole, and Hopkins 
(1938b) cases in which no preformed granules are associated with the 
vacuole, it is clear that the mitochondria are not necessary in all cases 
either for transport function or a support for enzymes. 

The supposed universality and permanence of mitochondria have led 
to many suggestions that they are concerned with some vital part of 
cellular activity, some function more universal than secretion and 
storage. Some evidence to this effect has been presented by Joyet- 
Lavergne (1927-35), both from the standpoint of the presence of 
materials active in respiration (see p. 121) and from the standpoint 
of a direct demonstration of respiratory activity. He finds that vital 
dyes are reduced most strongly near mitochondria and that individuals 
which have large amounts of mitochondria reduce the dyes faster than 
individuals with less mitochondria. He was able to demonstrate gluta- 
thione and vitamin A in the mitochondria and attempted to show that 
these two form an oxidation-reduction system. Rey (1931a, 1931b) 
repeated Joyet-Lavergne's staining experiments and obtained the same 
results, but criticized the latter's interpretation of his findings. Rey 
also repeated the experiments using an electrometric method for deter- 
mining rH and found no significant differences. Wurmser (1932) like- 
wise criticized Joyet-Lavergne's interpretations of the stain reactions as 
indications of oxidation-reduction differences. Bles (1929) found that 
the oxidation-reduction reactions which he studied in Arcella were asso- 
ciated with the hyaloplasm, rather than with any granules. Since Joyet- 
Lavergne found morphological continuity between the mitochondria and 



126 CYTOPLASMIC INCLUSIONS 

two types of reserve granules, the existence of a secretory function is 
possible, and it is not necessary to invoke respiration in order to find 
a function for these mitochondria. The respiratory function must be 
regarded as unproved, either from the standpoint of the proof of the 
presence of materials which could act as an oxidation-reduction system, 
or from the standpoint of the direct measurement of localized oxidation- 
reduction potentials. At best mitochondria as morphological entities 
cannot be necessary for respiration, since many species lack them at one 
time or another in the life cycle, and since in other cases they can be 
eliminated experimentally without fatal results (Kirby, 1936). 

All the various types of evidence — staining reactions, composition, 
function, and tracing through the life history — show that mitochondria 
in the Protozoa do not form a homogeneous group, but are actually a 
heterogeneous assortment which are associated merely by their ability 
to segregate Janus green B or by even less specific staining reactions. No 
one type is found in all Protozoa, and in all cases which have been 
carefully studied mitochondria are not self-perpetuating but arise de 
novo at some time during the life cycle. 

The Vacuome Hypothesis 

According to the vacuome hypothesis as applied in the Protozoa, there 
are only two fundamental cytoplasmic components in the Protozoa — the 
chondriome and the vacuome, since the Golgi bodies and vacuome are 
merely different aspects of the same thing. The term vacuome was 
substituted for the earlier term segregation granule as an indication of the 
supposed homology between the neutral red bodies in animal cells and 
the vacuoles of plant cells. Volkonsky (1929 on), Kedrowsky (1931- 
33), Hall and his associates (1929 on), Lynch (1930), and others have 
upheld the general conclusion that the granules stainable with neutral 
red are identical with the Golgi bodies; but Kirby (1931), MacLennan 
(1933, 1940), Bush (1934), Kofoid and Bush (1936), Daniels 
(1938), and others have demonstrated many cases of neutral red 
granules which are not osmiophilic (for a more detailed discussion of 
this point, see p. 140). It should be pointed out that the acceptance 
of the vacuome hypothesis is by no means universal in the Metazoa or 
Metaphyta, according to Weier (1933) and Kirkman and Severinghaus 
(1938). 



CYTOPLASMIC INCLUSIONS 127 

The ability to segregate neutral red has been ascribed to a specific 
reaction of the dye with a single material in the granule, the process 
being called the "neutral red reaction" by Koehring (1930) and in- 
cluded as a part of the "ferment theory of the vacuome" by Kedrowsky 
( 1932b) . The substance involved is supposed to be a proteolytic enzyme, 
a conclusion based on the work of Marston (1923), who showed that 
these enzymes are precipitated by combination with neutral red, Janus 
green, and other azine dyes. Le Breton (1931) has reviewed the rather 
voluminous literature resulting from this suggestion and concludes that 
the reaction is not specific, since ordinary proteins (an important con- 
stituent of most segregation bodies) also are precipitated. From 
the standpoint of cytology, the theory fails to explain how neutral red 
and Janus green have such difi^erent staining reactions in the granules 
of living cells. Hopkins's ( 1938a) experiments with Flabellula show that 
precipitates are formed in vacuoles after either Janus green or neutral 
red. If Janus green was first used, and then neutral red added, a red 
precipitate formed around the original green one. "The small neutral red 
vacuoles are, then, the same as the vacuoles in which the condensation 
granules are formed, but it appears that Janus green B stains a different 
component of these vacuoles than does the neutral red." 

Not one factor, but many are responsible for the segregation of neutral 
red by cytoplasmic granules. The role of pH in neutral red staining has 
been demonstrated by Chambers and Pollack (1927) and Chambers and 
Kempton (1937), who showed that neutral red tends to go from an 
alkaline region to an acid region so that "segregation granules" would 
be those which are acid relative to the hyaloplasm. Kedrowsky (1931) 
demonstrated in Opalina that normally the segregation granules have 
this relationship with the hyaloplasm and that the staining reactions 
can be changed by altering the pW of the cytoplasm. He also showed 
that the granules will take up acid dyes in the presence of albumoses, 
which was confirmed by Volkonsky (1933) and included under the 
term "chromopexie." Since neutral red has long been known as a 
lipoid stain (Faure-Fremiet, Mayer, and Schaeffer, 1910) it is possible 
that this may play a role in the staining of bodies which contain lipoids, 
such as the dictyosomes of gregarinida and the digestive granules of 
Ichthyophthhius. From these brief examples it is clear that the segrega- 
tion of neutral red and other vital dyes is influenced by many internal 



128 CYTOPLASMIC INCLUSIONS 

and external factors and that it cannot be considered specific in the 
sense that it combines with a single definite substance. 

Since many factors are involved in the segregation of neutral red, 
it is to be expected that more than one type of granule will be revealed 
by the use of this and similar dyes. Conclusive evidence of this lack 
of specificity is furnished by several Protozoa in which two or more 
types of granules are able to segregate neutral red at the same time, i.e., 
under identical conditions. Dangeard (1928) stained two types of 
granules in Euglena with neutral red — the vacuome and the mucous ap- 
paratus. The latter group may be extruded to form a mucous envelope 
or mucous hairs — an interesting example of true external secretion, and 
comparable to the staining of secretion granules in Metazoa by neutral 
red. However, Dangeard rejected the mucous apparatus as vacuome 
because it retains the neutral red after the death of the cell, while the 
other granules — the true vacuome — do not. Finley (1934) found four 
different groups of granules which segregate neutral red in Vorticella: 
pellicular secretions, pellicular tubercles, thecoplasmic granules, and re- 
fractile granules. He was careful to control the staining to avoid over- 
staining, so that his results cannot be questioned on that ground. Bush 
(1934) found in Haptophyra two sets of granules which are discon- 
tinuous in size and distribution. Mast and Doyle (1935b) complete the 
picture by showing that three groups of granules stain in Amoeba: 
vacuole refractive bodies, refractive bodies, and blebs on crystals, and 
they proved experimentally that these bodies are different in origin and 
in fate. The experiments of Kedrowsky (1931) on Opalina show the 
reverse picture — under certain feeding conditions, the growing segre- 
gation vacuoles lose their ability to take up neutral red, just as they do 
in vertebrate eggs. Hall and Loefer (1930) showed that the granules 
in Euglypha may vary in the same specimen from pink to bluish red 
or red violet. 

Since neutral red stains several different groups of granules and also 
does not stain all stages of the same granule, it does not of itself reveal 
fundamental homologies, and it seems to me to be unjustifiable to group 
all of these bodies as vacuome or under any other catchall term. As a 
preliminary step toward an accurate classification of this group, the 
digestive granules — i.e., those neutral red bodies associated with the 
gastrioles — are separated from the segregation granules which cor- 



CYTOPLASMIC INCLUSIONS 129 

respond to the "vacuome de reserve" of Volkonsky and the segregation 
apparatus of Kedrowsky. The segregation granules of necessity still 
include a heterogeneous assortment and probably include granules other 
than those associated with synthesis and storage, but at present they 
cannot be classified properly because too many of them are known only 
by their ability to segregate neutral red. Kedrowsky and Volkonsky 
regard both types as carriers of enzymes, in one case acting to digest 
proteins and in the other case to synthesize them. However, each group 
appears de novo when the necessity arises, and the two groups show 
no direct continuity with each other. Further evidence of the independ- 
ence of these two groups of granules is furnished by Opdina, in which 
only the segregation bodies are present, and by Ichthyophthir'ms, in which 
only the digestive granules are present, these latter having no connection 
with the storage of the numerous protein granules. 

Digestive Granules 

The digestive granules may be briefly defined as cytoplasmic granules 
stainable with neutral red, which become associated with the newly 
formed vacuoles containing food. This does not include all neutral 
red granules in the food vacuole, since such granules as the vacuole 
refractive bodies of Amoeba are derived from the food (Mast and 
Doyle, 1935b) and thus are not cytoplasmic components. Volkonsky 
(1934) points out that the term vacuole has been applied to so many 
structures that it is a source of confusion, and he has substituted the term 
gastriole. The fluid vacuole which contains ingested food is a pro- 
gastriole, and with the addition of the digestive granules (vacuome in 
Volkonsky's terminology) becomes a gastriole. In addition to these 
terms it is convenient to use postgastriole for the structures containing 
undigested remnants. 

There is no single pattern of the gastriole in the Protozoa. In 
A. proteus, mitochondria alone aggregate periodically around the gas- 
triole (Mast and Doyle, 1935b); in Paramecium and Campanella, the 
digestive granules enter the gastriole, and mitochondria cluster around 
the membrane in the alkaline phase; in Flabellula, the materials stain- 
able with Janus green or neutral red are normally dissolved in the fluid 
vacuole, which later forms the gastriole (Hopkins, 1938), and a some- 
what similar situation is found in hypermastigote flagellates (Duboscq 



130 



CYTOPLASMIC INCLUSIONS 



and Grasse, 1933); in Ichthyophthirius (MacLennan, 1936), the diges- 
tive granules (Fig. 31) enter the gastriole, but the mitochondria are 
never associated in any visible manner with the digestive mass. A more 
detailed description of digestion will not be given in this section, since 
attention is here centered on the granules. 

All careful descriptions agree that the digestive granules vary in 
size and shape during the gastriolar cycle, ranging from minute spherules 
to relatively large rods. Both vital stains and metallic impregnation show 
a homogeneous structure, and the deformation of these granules either 












I 



Figure 31. The association of the gastrioles and the digestive granules in Ichthyoph- 
thirius multifiliis. (From MacLennan, 1936.) 



from other cytoplasmic granules or from outside pressure show that 
they have a soft, semifluid consistency. Volkonsky (1934) has found 
that the morphology of the gastriole varies in the same species with the 
food used. In Acanthamoeha he was able to induce the formation of 
large granules, small granules, or a homogeneous rim stainable with 
neutral red, by varying the food used. No digestive granules were formed 
around vacuoles which contained only starch. 

The penetration of the digestive granules into the vacuole was not 
observed by Koehring (1930), Hall and Dunihue (1931), Dunihue 
(1931), or Hall and Nigrelli (1930), and the suggestion was made 
that the granules in the food vacuole are derived from food particles 



CYTOPLASMIC INCLUSIONS 131 

rather than from the cytoplasm, a suggestion which later was proved to be 
true, in the case of vacuole refractive bodies of A. proteus, by Mast 
and Doyle (1935b). Volkonsky, however, checked his observations on 
many ciliates and ruled out exogenous granules in the gastrioles of 
Glaucoma by the use of bacterial free medium; he still observed diges- 
tive granules in the vacuoles and was able to trace them from the cyto- 
plasm. The migration of the digestive granules into the gastriole of 
Ichthyophthirius was found by MacLennan (1936), but it was shown 
that the granules do not penetrate any membrane. Instead, a new mem- 
brane is formed (Fig. 31) around the whole gastriole and the inner 
membrane, which is the original one, then disappears. The end result 
is exactly the same as in the cases described by Volkonsky, but the 
mechanism is somewhat different. 

Volkonsky's interpretation of the digestive granules as vacuome is 
based upon their impregnation by the various Golgi-type methods. The 
question as to the identification of the granules as shown by entirely 
different methods is not present in this case, as it was in the case of the 
scattered cytoplasmic granules (segregation granules), since the digestive 
granules can be recognized independently of their staining reactions by 
their relationship to the gastriole. Hall and Nigrelli (1937) claim that 
the digestive granules are less consistent in impregnation than the 
scattered cytoplasmic granules and dispute Volkonsky's claim that they 
can be considered as vacuome. MacLennan (1940) showed that the 
various types of osmiophilic granules could not be distinguished on the 
basis of impregnation alone; in particular the digestive granules of 
Ichthyophthirius show 100-percent impregnation. 

Few of the materials which occur in the digestive granules are known 
from direct evidence. The digestive granules of Paramecium are high 
in ash (MacLennan and Murer, 1934) and those in Ichthyophthirius 
contain lipoids (MacLennan, 1936). These latter bits of information 
are not as yet particularly useful and emphasize the need for more 
specific knowledge. The presence of enzymes is suggested by the morpho- 
logical evidence and this would be a fertile field for the use of the 
microenzymatic methods. 

The digestive granules are not permanent self-perpetuating structures, 
but appear to rise in the cytoplasm in response to the stimulus of feeding. 
Volkonsky (1934) found that when the preexisting granules are utilized 



132 CYTOPLASMIC INCLUSIONS 

in the formation of gastrioles, new granules are formed in the cyto- 
plasm. The digestive granules in Ichthyophthmus were observed by 
MacLennan (1936) to arise de novo in direct contact with the ingested 
particles of food. Final evidence of their de novo origin was furnished 
by the fact that no digestive granules are present in the encysted stage, 
after the gastrioles which were formed during the feeding stage have 
disappeared. Volkonsky suggests that the materials of the digestive 
granule exist in the cytoplasm in a diffuse state. In the hypermastigote 
flagellates this material is concentrated in dissolved form in the gastrioles; 
in Flabellula the materials are first concentrated in vacuoles, which con- 
tribute the fluid part of the gastrioles; in the more common cases, the 
materials are condensed to form digestive granules. We must not for- 
get, however, that not all the digestive reactions result in granules, since 
dipeptidase in Amoeba is independent of any granules. Whatever the 
specific morphology of the reaction, whenever the cytoplasmic equilib- 
rium is disturbed by the ingestion of food there is an effective mobiliza- 
tion of this material to cope with the ingested food. Volkonsky calls 
these varied changes the "vacuolar reaction," an extremely useful term, 
but since we reject the term vacuome, "gastriolar reaction" would be 
more appropriate. 

Segregation Granules 

The segregation granules are bodies which are able to concentrate, 
accumulate, and store within themselves vital dyes, proteins, and other 
materials. Unlike the definitions of most cytoplasmic granules, this 
definition is based upon function rather than upon morphology or 
staining reactions. The evidence for this definition is due largely to the 
work of Kedrowsky (1931-33) on Opalina, Spirostomum, and other 
ciliates. Since the functions of most of the granules stainable with 
neutral red have not been demonstrated, the work on Opalina will be 
discussed first and granules in other Protozoa considered in the light of 
this work. The accumulation of vital dyes in higher concentration than 
they occur in the medium is, of course, one piece of evidence of the 
segregating ability, even if this has not been demonstrated with the 
materials which enter into the metabolism of the cell. 

The segregation bodies of Opalina (Figs. 32-35) are the external 
layer of granules or ectosomes and have been identified as Golgi bodies 



CYTOPLASMIC INCLUSIONS 133 

or as mitochondria by various authors, since they respond to some of 
the Golgi and mitochondrial techniques, although they are not stained 
specifically with Janus green B. Kedrowsky described four main morpho- 
logical types — fine dispersed granules, large granules, alveoli, and 
heteromorphic granules (Figs. 32-35). In some cases the dispersion of 
granules is so accentuated that they lose their identity as granules. These 
changes in morphology are associated under natural conditions with the 
seasons of the year; for instance, the heteromorphic types are common 
in the spring and early summer, and the large granular type is found 
in the early spring. There is some variation between populations of 
different frogs in the same season, but all members of the same popula- 






52 




?t 



33 ^ 35 



Figures 32-35. Basic morphological variations in the segregation granules of Opalina 
ranarum. Semischematic. (After Kedrowsky, 1931e): Figure 32. Dispersed type; Figure 
33, homomorphic granular type; Figure 34, alveolar type; Figure 35, heteromorphic 
granular type. 

tion have essentially the same type of granules. These different forms 
can be produced experimentally by changing the culture medium. The 
colloidally dispersed type is typical of amino-acid cultures. In distilled 
water, each ectosome swells and becomes a watery vacuole. The hetero- 
morphic type is found in cultures which contain defibrinated and hemo- 
lysed blood. These changes, which occur both naturally and in artificial 
media, may well account for the disagreement among cytologists both 
as to the morphology and the identification of these granules. 

The segregation bodies of Opalina are not permanent organelles. As 
indicated above, they may disperse homogeneously through the ecto- 
plasm, which then takes a general pale stain with neutral red, and no 
method applied at this stage shows any indication of a remnant of the 
originally discrete granules. If the bodies are loaded with protein com- 
pounds of metals, such as silver, they may finally be extruded from the 
surface and be replaced by granules which arise de novo. 



134 



CYTOPLASMIC INCLUSIONS 



The composition of the segregation bodies is as varied as their 
morphology and results from the same causes — changes in the environ- 
ment. Under natural conditions they may contain proteins (Millon's 
reaction), glycoproteins (Fischer's reaction), cholesterin (Schultz re- 
action, digitonin reaction), or be stained with bile pigments. The 
alveolar type of course contains mostly water. The segregation granules 
will store basic dyes in salt solution, acid dyes in the presence of pro- 
teins, silver in the form of kollargol or other similar compounds, iron 
albuminates, cholesterin, and so forth. According to the "ferment theory 
of the vacuome" of Kedrowsky (1932b), enzymes are present in the 





Figures 36-37. Segregation granules in Trypanosoma diemyctyli, neutral red stain. 
(After Nigrelli, 1929.) Figure 36. Preformed granules around the blepharoplast; Figure 
37, preformed granules and granules induced by prolonged exposure to neutral red. 



segregation bodies, but, as is the case with the digestive granules, no 
direct proof of this is available. 

The segregation bodies obviously function in the concentration and 
storage of various materials, particularly proteins. This may be accounted 
for on a purely physical basis, as, for instance, the tendency of basic 
dyes to migrate to a more acid region, or the tendency of molecules to 
migrate toward bodies of opposite charge. The exact mechanism is, of 
course, a complex problem and cannot be taken up here, but the es- 
sential point at present is that the granules may play a purely passive 
role — the accumulation and storage of materials which originate else- 
where. 

Accumulation is merely one part of the function, according to the 
enzyme hypothesis of the segregation granules, advanced at nearly the 
same time in slightly varying forms by Koehring (1930), Kedrowsky 
(1931 on), and Volkonsky (1929 on). Koehring based her conclu- 
sions on the supposed specificity of the neutral red reaction for proteo- 
lytic enzymes (see p. 178). Kedrowsky considers that both the synthesis 



CYTOPLASMIC INCLUSIONS 135 

of proteins and deaminization may occur in the segregation bodies. The 
evidence of synthesis may be summarized simply as the increase in size 
or number of segregation bodies in OpaVma when immersed in various 
nutrient solutions, and the subsequent identification in the granules of 
materials from this culture medium. This certainly proves the segregat- 
ing ability, but the actual synthesis might take place almost anywhere 
and the increase in size be due simply to the segregation of these pre- 
formed materials. The evidence of deaminization is based on the appear- 
ance of glycoprotein in the late stages of the segregation bodies and 
the fact that the segregation granules are able to oxidize Rongalit white 
vitally. This evidence could hardly be called more than suggestive, but 
the processes which are indicated by these tests seem to be localized in 
the segregation granules. 

The neutral red granules of Protozoa other than Opalina, to be con- 
sidered in the rest of this section, excludes only the group which were 
discussed above as digestive granules. It is thus essentially the group 
called segregation apparatus by Kedrowsky (1931), or the vacuome of 
Hall (1929 on). Since neither the history nor the function of most of 
these granules is known, this is doubtless a heterogeneous group, but 
I believe that further splitting at this time would merely add names 
without increasing understanding. 

The segregation bodies are normal cytoplasmic constituents and are 
not induced by vital dyes, since they have been observed by many in- 
vestigators in normal, unstained specimens (Hall, 1929 on; Finley, 
1934; MacLennan, 1933, 1936; Volkonsky, 1929 on; Kedrowsky, 1931 
on, and others). Prolonged staining (Fig. 23), it is true, may induce 
the formation of new granules (Kedrowsky, 1931; Cowdry and Scott, 
1928; Nigrelli, 1929), but this is not a universal phenomenon, since 
many species, in my own observations, show the general diffuse stain- 
ing of the cytoplasm and nucleus characteristic of severe overstaining, 
without the appearance of new granules. The normal origin of all types 
of segregation granules seems to be de novo. Mast and Doyle (1935b) 
removed most of the refractive bodies from A. proteus and observed the 
formation of new bodies several hours after feeding. The ""blebs" on 
the crystals likewise clearly originate de novo. These cases are too few 
to justify any certain statements for all the many segregation granules, 
but it is indicative that in all the many descriptions of these granules, 



136 CYTOPLASMIC INCLUSIONS 

there has been no clear case of division described — all the evidence indi- 
cates a de novo origin. 

Small homogeneous spherules are the commonest type of segregation 
body and they are found in all classes of Protozoa. There are often more 
than one group in a single individual, as shown by differences in size 
(Bush, 1934) or differences in localization (Finley, 1934). Complex 
bodies similar to those found by Kedrowsky in Opalina have been found 
in flagellates from termites (Kirby, 1932). In some cases such complex 





Figure 38. Segregation granule (refractive body) of Amoeba proteus in successive 
stages of resorption. (From Mast and Doyle, 1935.) A, normal body; B-F, stages in 
resorption, optical sections ; D, surface view. Outer layer cross-hatched, shell black, 
vacuole in outline. 

bodies may be the result of precipitation of dyes or other materials in 
an aqueous vacuole, but in the cases of the denser granules entirely^ 
different methods show the duplex structure. In the refractive bodies 
of Amoeba (Fig. 38), microchemical tests show that the duplex struc- 
ture represents an actual difference in composition (Mast and Doyle, 
1935a). 

The segregation granules are most often scattered at random through- 
out the endoplasm (Hall and his associates, 1929 on), but occasionally 
are more definitely localized. In Trypanosoma. (Figs. 36, 37) they are 
concentrated around the blepharoplast (Nigrelli, 1929), in the Ophry- 
oscolecidae they are most common in the operculum and spines (Mac- 
Lennan, 1933), and in Lechriopyla a marked concentration is found just 
under the pellicle (Lynch, 1930). In Vorticella a group of discrete 
globular inclusions is found scattered in the cytoplasm, and another group 
is found in the stalk (Finley, 1934). The tubercles of the pellicle also 
stain, as do the secretions of the pellicle, but the latter have been consid- 
ered in the section on secretion granules and the former do not seem to 
me to fulfill the definition of cytoplasmic granules, or at least are clearly 
not in the same class as the ectosomes of Opalina. As a contrast to these 
Protozoa with several types of segregation granules, the ciliate Ichthy- 



CYTOPLASMIC INCLUSIONS 137 

ophthirms has none at all at any time in its life cycle (MacLennan, 
1936), and the function of synthesis and storage of the protein re- 
serves is taken over by the macronucleus. 

The composition of the segregation granules of Protozoa other than 
Opalina is known only in the case of the refractive bodies and blebs 
of Amoeba. The osmiophilic shell of the refractive bodies of Amoeba 
has a protein stroma impregnated with a lipid substance (Mast and 
Doyle, 1935a). This portion stains with Sudan III only after Ciaccio's 
method for "unmasking" the lipoids, and is intensely blue in Nile blue. 
These granules are not dissolved in alcohol in twelve hours, but they 
do lose their positive reaction to fat soluble dyes. They respond to the 
methylene-blue-sulphuric test for metachromatin and give a faint re- 
action with Millon's reagent. Within this layer a brittle carbohydrate 
shell and an unknown fluid are found. The stainable blebs on crystals 
in the same protozoan, when first formed, contain only lipoids, but as 
they grow larger, protein is added so that their final composition is 
similar to the shell of the refractive bodies of the cytoplasm. These 
granules are therefore quite different from the segregation granules of 
Opalina, since Kedrowsky showed that whatever else might be stored 
in these bodies, they do not segregate lipoids. However, since the re- 
fractive bodies do segregate proteins and stain with neutral red, they 
are included as segregation bodies. This is, of course, arbitrary, since 
they overlap on the Golgi granules and on the reserve granules. Suffi- 
cient mineral ash is present to mark the vacuome in incinerated speci- 
mens of Opalina (Horning and Scott, 1933) and Paramecium (Mac- 
Lennan and Murer, 1934). The only striking fact about the segregation 
bodies in most Protozoa is that with the exception of those in Opalina and 
Amoeba, not even a sketchy outline of their composition is available. 

The function, in so far as it is known, agrees closely with the storage 
function described for the granules in Opalina. Variations of the num- 
bers of the segregation granules have been found in Paramecium (Duni- 
hue, 1931) and in Vorticella (Finley, 1934). Dunihue was able to 
correlate the decrease in numbers with starvation, thus indicating a 
storage function. Mast and Doyle (1935a) showed that both protein and 
lipoid materials are found in the blebs on the crystals and on the re- 
fractive bodies. Since the blebs appear shortly after feeding and since 
they in turn disappear as the refractive bodies are increasing, the blebs 



138 CYTOPLASMIC INCLUSIONS 

seem to be temporary reserves, while the refractive bodies are the final 
reserves. In the blebs, the protein portion appears later than the lipoid 
portion. There is no evidence in these cases of anything more than the 
accumulation of materials. 

GoLGi Bodies 

The term Golgi body has been applied in Protozoa to organelles 
which differ fundamentally in both composition and function. These 
structures include contractile vacuoles (Ramon y Cajal, 1904-5), gran- 
ules (Hirschler, 1914), specialized regions of cytoplasm around the 
contractile vacuoles (Nassonov, 1924), segregation granules (Cowdry 
and Scott, 1928), osmiophilic nets (Brown, 1930), and the parabasal 
apparatus (Duboscq and Grasse, 1925). The controversies which have 
arisen between advocates of one or another of these structures have 
been due not so much to disagreement upon the actual facts involved as 
to disputes concerning criteria for the identification of Golgi material. 
The selection of criteria is thus a crucial point in coordinating the in- 
vestigations of Golgi bodies; yet even after years of work on representa- 
tives of all the major groups of animals and plants, and notwithstand- 
ing periodic reviews of the field, few criteria seem to have unanimous 
approval — few, indeed, have majority approval. The most recent re- 
view (Kirkman and Severinghaus, 1938) after failing to demonstrate 
any universal and objective basis of identification, quotes as follows 
from Gatenby (1930): "modern workers in general have experienced 
no difficulty in identifying Golgi bodies." This is very satisfactory as 
long as only one school of cytologists is considered, but such state- 
ments lose their attractive ring of authority when one tries to correlate 
the results presented by such experienced cytologists as Bowen, Parat, 
Gatenby, Canti, or Ludford, to mention only a few. 

This is particularly true when seeking criteria on which to base a 
reasonable identification of Golgi bodies in the highly specialized cells 
of the Protozoa. In the following paragraphs the major objective cri- 
teria which have been used are discussed with particular reference to 
their applicability to the Protozoa. In general these criteria involve two 
points — consistent impregnation, and occurrence in all types of cells. 

The first Golgi structures were discovered by the use of metallic im- 
pregnation methods, and ever since these methods have remained as 



CYTOPLASMIC INCLUSIONS 139 

the primary criteria (Gatenby, 1930), particularly since the discovery 
that the supposedly typical reticular network is a relatively rare type 
and in many cases is due to a temporary aggregation of granules (Hirsch- 
ler, 1927) . Both the osmic and the silver methods involve the same prin- 
ciple — the adsorption of the reduced metal on particular structures, 
although the actual reduction of the metallic compound may take place 
either where the deposits are located or in other parts of the cell (Owens 
and Bensley, 1929). Since the osmic acid methods have been most 
thoroughly investigated, this discussion will be restricted to these tech- 
niques, but the fact should be emphasized that the same general principles 
are involved in the silver techniques as well. 

The preliminary treatment of the cell strongly affects the subsequent 
impregnation (Lison, 1936). Thus direct exposure of the protozoan 
to osmic fumes (Hall, 1929) is not equivalent to the full technique 
which involves preliminary fixation in such mixtures as Champy's fluid. 
Likewise, the blackening of granules by exposure to osmic fumes after 
they have been stained with neutral red is not the equivalent of the 
standard Golgi methods, since it has been shown in the case of Flabel- 
lula (Hopkins, 1938b) that the blackening is produced only in the 
presence of neutral red. 

The Golgi methods are not specific for any single type of material, 
since a series of different lipoids extracted from echinoderm eggs give 
a typical Golgi reaction (Tennent, Gardiner, and Smith, 1931). Further- 
more both lipoid and non-Iipoid bodies of various types in the Protozoa 
react typically and identically to the Golgi techniques (MacLennan, 
1940). Although the method is not specific in the sense that it reveals 
a single known substance, or some unknown Golgi material, the method 
is consistent in the sense that under proper conditions the results can 
be reproduced. In general, the reputation of osmic acid methods as 
carpricious and erratic is due to the slow penetration of tissues by 
osmium tetroxide, with the result that the cells in a block vary in ex- 
posure to the unreduced reagent because of their varying distances from 
the free surfaces. Another source of difficulty is the fact that the com- 
position of cytoplasmic granules changes during the growth of that 
granule (Kedrowsky, 1931e; Mast and Doyle, 1935a, 1935b; Mac- 
Lennan, 1936), and it is to be expected that the reducing power will 
also vary. The Protozoa are admirably adapted to solve both of these 



140 CYTOPLASMIC INCLUSIONS 

difficulties. The individual cells are either separate or in such small 
aggregates that all cells and all parts within the cell are exposed to 
essentially the same concentration of reagents. The variations in the 
stage of the Golgi granules can be observed directly in living Protozoa, 
so that the impregnation of the granules can be compared at equivalent 
stages, which can be identified with or without impregnation. In meta- 
2oan cells the Golgi nets are extremely difficult to demonstrate in the 
normal living cell, so that it is at present impossible in these cells to 
check the structures independently of impregnation. When standard 
conditions are achieved both around the cell and within the cell, abso- 
lutely consistent results are attained, even with osmic acid (MacLennan, 
1940). Under these conditions, 100 percent of the digestive granules, 
fatty acid granules, excretory granules, and so forth, impregnate, with 
the result that these types cannot be separated on the basis of the Golgi- 
type reactions. The results of Hall and Nigrelli (1937), which seem 
to show that excretory granules and digestive granules are erratic in 
impregnation, is due to failure to allow for changes in composition and 
aggregation or for the occasional lack of these granules at certain times 
in the life cycle. All the stages have been lumped together, rather than 
equivalent stages compared. 

In view of the fact that osmic acid is reduced by many different sub- 
stances, it is highly important to prove that the impregnated bodies 
are not some other component (Hirschler, 1927; Bowen, 1928). Re- 
sistance to bleaching by turpentine or hydrogen peroxide is more pro- 
nounced in Golgi bodies than in most mitochondria, but unfortunately, 
even with this method, there are too many border-line cases in which 
the individual judgment of the observer is the determining factor. 
However, this individual judgment can be eliminated in comparing 
Golgi bodies and mitochondria by the use of such methods as the Alt- 
mann aniline acid fuchsin stain after osmic impregnation. Such a com- 
parison of Golgi bodies and segregation granules is not possible, since 
the major criteria of the one is a fixation method and of the other 
is a vital staining method. If the two components have a different 
distribution, a comparison of different cells is sufficient to establish the 
difference (MacLennan, 1933), but too often there is in both cases 
merely a random distribution. The most famous case of this sort is that 
of the gregarines, in which Joyet-Lavergne (1926b) described the Golgi 



CYTOPLASMIC INCLUSIONS 



141 



bodies as being identical with the neutral red bodies because of simi- 
larity in form and distribution, and this has been widely accepted in 
spite of objections presented by Tuzet (1931) and Subramaniam and 
Ganapati (1938). The question was not settled conclusively until 
Daniels (1938) applied the centrifuge to these species. She found 
that Golgi bodies always moved to the centripetal pole of the cell, but 



39 






# 

■>ij:: 



40 



. o 

41 







43 



Figures 39-45. Dictyosomes: Figure 39, Dictyosomes from Haptophrya tnichiganensis, 
Champy-osmic (after Bush, 1934) ; Figure 40, diagram of a dividing dictyosome of 
Lecudina brasili (after Subramanian and Ganapati, 1938) ; Figure 41, stages in the 
secretion of neutral fat in Ichthyophthirius multifiliis, Lorrain Smith Nile blue-sulphate 
method, black represents blue stain, stippling represents pink (after MacLennan, 1934) ; 
Figures 42-45, dictyosomes during the life cycle of Lecudina brasili; Figures 42 and 43, 
intracellular stages; Figure 44, growing trophozoite; Figure 45, "association" stage, 
either daFano or Nassonow impregnations (after Subramanian and Ganapati, 1938). 

that the bodies stainable with neutral red were never displaced. Thus 
in this case the Golgi bodies and the neutral red granules are not 
identical. This does not mean that no osmiophilic granules segregate 
neutral red, since, for example, the digestive granules react to both 
impregnation and vital staining, but it does mean that the ability to 
segregate neutral red is not a characteristic of all the Golgi bodies of 
the Protozoa. 



142 



CYTOPLASMIC INCLUSIONS 



The theory that the Golgi apparatus is a universal organoid of the 
cell, as constant in its characteristics as is the nucleus, has given rise 
to a series of criteria requiring permanence during the whole cycle of 
the cell, as well as similarity in form and intracellular distribution. The 
presence of Golgi bodies in all stages of the life cycle has been demon- 
strated in Sporozoa (Joyet-Lavergne, 1926a) as well as their origin 
by the division of preexisting Golgi bodies (Subramaniam and Gana- 
pati, 1938, Figs. 42-45). However, this is not universal, since neither 





Figures 46-47. The effect of centrifuging upon the distribution of cytophismic 
granules. Figure 46, diagram of a centrifuged gregarine, F, fat, GAa granular Golgi 
material, GAb larger Goli elements, N nucleus, K karyosome, M mitochondria, P 
paraglycogen, C "chromidia," neutral red bodies not shown; the paraglycogen mass 
marks the centrifugal pole. (From Daniels, 1938.) Figure 47, contractile vacuole of 
a centrifuged amoeba, v vacuole, b mitochondria ; the mitochondria are thrown to the 
centrifugal surface of the vacuole. (From Mast and Doyle, 1935b.) 

of the types of Golgi bodies in Ichthyophthirius ( MacLennan, 1936) 
nor Amoeba (Mast and Doyle, 1935a) are self-perpetuating or even 
present in all stages of the cycle, but arise de novo. Thus the Golgi 
bodies are not universally self-perpetuating and permanent. 

The criterion of similarity in form has received considerable support, 
but the evidence as to what this form is has been discordant. Nassonov 
(1924) described a net-like structure around contractile vacuoles and 
homologized this with the Golgi net. Hirschler (1927) finds that the 
typical Golgi bodies have an osmiophil cortex and an osmiophobe 
center, this duplex structure being called a dictyosome. The complex 
nets around the contractile vacuoles are, according to Hirschler, aggre- 



CYTOPLASMIC INCLUSIONS 143 

gations of the dictyosomes. Subramaniam and Ganapati (1938) insist 
on this dictyosome structure, although they describe a homogeneous 
spherule in one stage of the Golgi cycle: 

a Golgi granule when it enlarges becomes differentiated into a vesicle having 
chromophile and chromophobic regions. Rupture of the vesicle gives rise to 
batonettes in which the chromophobic part is in relation with the cytoplasm. 

Since all of these structures — spherules, dictyosomes, aggregations — may 
be included in the cycle of a single granule (Kedrowsky, 1931; MacLen- 
nan, 1936; Mast and Doyle, 1935a) or as the result of the periodic ag- 
gregration of granules (Hirschler, 1927; MacLennan, 1933), it is 
impossible to insist on one type to the exclusion of the others, and we 
must conclude that morphology is not a criterion for the identification 
of various types of granules. 

Similarity in distribution within the cell has also been urged as a 
criterion. Hall (1931) holds that a random distribution throughout the 
cytoplasm is the typical configuration, while Subramaniam and Gopala- 
Aiyar (1937) consider that an excentric juxtanuclear position, similar 
to that found in spermatids or gland cells, is typical. In metazoan cells 
both types have been found, and both may be typical of the same cell if 
all stages of the life q'cle are considered. Similar periodic aggregations 
and dispersal of Golgi granules have been described in connection with 
the pulsatory cycle of the contractile vacuoles of ciliates (MacLennan, 
1933, 1936). These movements are comparable to the migration of 
mitochondria and of digestive granules, which have likewise been as- 
sociated with functional changes in the cell. 

The chief characteristic either of form or of distribution of Golgi 
bodies is a variability which is associated with functional changes, and 
even this variability is not a criterion since it is characteristic of all gran- 
ules which are actively concerned with the metabolism of the cell. 

The criteria based upon impregnation and upon separation from other 
granules which can be identified by more specific methods are the only 
truly objective criteria, while the criteria of universality in form, dis- 
tribution, and permanence are indefinite and are based upon various 
theories of the function, form, or derivation of Golgi bodies. The term 
Golgi body, being based upon nonspecific criteria (nor would retention 
of specific form or distribution make the definition more exact), in- 



144 CYTOPLASMIC INCLUSIONS 

eludes a heterogeneous group of structures, including scattered endo- 
plasmic granules and granules associated with contractile vacuoles or 
fused to form heavy and permanent vacuolar membranes. A summary 
of the known functions of these granules includes the excretion of ma- 
terials through the contractile vacuoles, the formation of granules of 
neutral fat, the storage of lipoids other than neutral fat, a secretory cycle 
which does not involve lipoids, as well as many functions as yet unknown. 
These functions are different, but in a broad sense they are all varieties of 
secretion and in this respect conform to the Nassonov-Bowen theory of 
the relationship between Golgi bodies and secretion. The restriction of 
criteria for Golgi bodies to that of impregnation alone thus does not do 
violence to the concept of Golgi bodies as originally developed in verte- 
brate tissues. 

The Golgi bodies are simply those secretory bodies (exclusive of 
mitochondria and segregation granules) which synthesize or store ma- 
terials which can be preserved by Golgi-type fixatives, and after this 
treatment are able to reduce (or adsorb the reduced metal) OSO4 or 
silver nitrate. This is not a natural grouping, since on the one hand it 
includes several specific types of secretion, and on the other hand it does 
not include all types. In several cases the Golgi bodies can be classified 
according to function or composition, and in this discussion they are 
referred to as excretory granules, intermediate lipoid bodies (see p. 
151), and, when the bodies are secretory in nature but the type of 
secretion body unknown, secretory Golgi bodies. This leaves a miscel- 
laneous group of Golgi bodies which are known only by their ability 
to reduce osmium or silver and for which there is no evidence as to 
either composition or function. Since the impregnation reactions them- 
selves do not reveal homologies which must be based on composition 
and function, the retention of the term Golgi body is merely a con- 
venience to bridge the change from reliance on the nonspecific osmic 
techniques alone to reliance upon specific cytochemical and physiological 
criteria. 

Excretory Granules 

Ramon y Cajal (1903-4) was the first to suggest that the contractile 
vacuole is equivalent to the Golgi reticulum of the cells of the Metazoa. 
The first confirmation of this view was the demonstration in several 
ciliates by Nassonov (1924) that this vacuolar region is osmiophilic. 



CYTOPLASMIC INCLUSIONS 145 

Further work has extended the number of such cases in cihates and 
flagellates, but at the same time it has been definitely proved that the 
vacuoles in several species of both classes are never osmiophilic. An 
examination of these cases shows that they form a closely graded series 
ranging from no impregnation at any time to complete impregnation at 
all times. Fabrea has no ectoplasmic Golgi bodies and no osmiophilic 
contractile vacuoles (Ellis, 1937). Lechriopyla has many ectoplasmic 
Golgi bodies, but they never form an aggregation around the contractile 
vacuole (Lynch, 1930). Epid'mhm, Eudiplodinium (Fig. 54), and oth- 
ers show an accumulation of granules only during diastole (Kra- 
scheninnikow, 1929; MacLennan, 1933). This same type is found in 
IchtJjyophthirms in the parasitic stages (Fig. 48), but neither free osmio- 
philic ectoplasmic granules nor accumulations around the contractile 
vacuoles during encystment (MacLennan, 1936) are present. Meta- 
d'm'ium (Fig. 55) has a permanent granular nephridioplasm which 
waxes and wanes during the pulsatory cycle (MacLennan, 1933). Va.ra- 
mecium caudatum and P. nephridiatiim have a permanent osmiophilic 
shell around the radiating canals, but not around the contractile vacuole 
itself (Nassonov, 1924; von Gelei, 1928) . Haptophrya possesses a vacuo- 
lar apparatus which consists of a permanent, homogeneous osmiophilic 
tube (Bush, 1934). This nicely graded series shows that the impregna- 
tion of parts of the vacuolar apparatus is due to aggregations of osmio- 
philic granules around the fluid vacuoles and their membranes. The 
only cases in which the membranes themselves impregnate are those ex- 
tremely specialized cases in which the osmiophilic material forms a 
permanent shell around the fluid vacuole. The highly complex osmio- 
philic apparatus in either Paramecium or Haptophrya is fundamentally 
no different from those vacuoles with a granular layer, and the homo- 
geneous osmiophilic shells are merely the result of the aggregation and 
specialization of the ordinary undifferentiated ectoplasmic Golgi bodies. 
This view is similar to that of Nassonov (1924) and Hirschler (1927), 
except that these authors view the osmiophilic portion as the outer por- 
tion of the dictyosomes, and the fluid vacuole as the inner portion of the 
dictyosome structure. Both views are, of course, the same, if the osmio- 
phobic portion of the dictyosome be accepted as a secretion droplet and 
not as an essential part of the dictyosome itself, as held by Gatenby and 
Subramaniam and Ganapati. 



146 



CYTOPLASMIC INCLUSIONS 



The demonstration that the osmiophiHc granules are an addition to 
the simple contractile vacuoles which may exist independently of the 
granules, even in the same species, is a close parallel to the union of 
the progastriole and digestive granules. The only difference is that no 
gastriole yet discovered presents a permanent, highly developed mem- 



O 








48 




O (^ 



so 



51 




Figures 48-52. Excretory granules and contractile vacuoles. Figure 48, aggregation 
and disappearance of excretory granules during the pulsatory cycle, from Ichthyophthirius 
multlfilih, Champyosmic impregnation (after MacLennan, 1934) ; Figures 49-50, from 
Polyplastron tntdtivesiculatuiyi : Figure 49, cold impregnation; Figure 50, warm im- 
pregnation (after MacLennan, 1933) ; Figure 51, from Dogielella sphaerii, Champyosmic 
(after Nassonov, 1925) ; Figure 52, "nephridialplasm of dvnpanelLj lunbellaria, 
Flemming-glychemalum (after Faure-Fremiet, 1925). 

brane or a permanent granular region, as in the contractile vacuoles of 
Metadhiium , Pavamec'ium, and Haptophrya. 

The origin of a new vacuolar apparatus from the original structure of 
the parent has been described and compared with dictyokinesis in germ 
cells (Nassonov, 1924; von Gelei, 1928). The clearest case is that of 
Haptophrya, in which the vacuolar apparatus is a tube extending the 
full length of the ciliate. Studies of both live and fixed animals show 
that this tube is permanent and that the transverse fission of the cells 



CYTOPLASMIC INCLUSIONS 147 

divides the tube in two parts, each of which continues to function in the 
daughter cells (Bush, 1934). The only possible exception would be the 
stages in which the parasite is transferred from one host to another. The 
new vacuolar apparatus of Paramecium is said to arise by the multipli- 
cation of canals and by the division of the whole vacuolar apparatus just 
prior to fission (Nassonov, 1924). The formation of extra vacuoles has 
been noted many times in living ciliates, but there is no recorded observa- 
tion of the actual division in a living Paramecium, and the interpretation 
of Nassanov's figures of fixed material is susceptible to the difficulties 
inherent in building any cycle from fixed material alone. 

The majority of vacuolar systems, however, do not possess the thick, 
permanent wall similar to that in Haptophrya, but a temporary aqueous 
vacuole which certainly arises de novo (Taylor, 1923; Day, 1927; Mac- 
Lennan, 1933, 1936). These vacuoles and their membranes are not 
osmiophilic, the impregnation of the vacuolar system being due to the 
aggregation of the osmiophilic excretory granules. The fundamental 
question with respect to the origin of Golgi bodies is in these cases not 
the origin of the vacuoles, but the origin of the individual excretory 
granules. These granular aggregations in the Ophryoscolescidae have 
been observed in living specimens (Figs. 53-55) to be ectoplasmic Golgi 
bodies which migrate into the region of the vacuole during systole and 
the earliest stages of diastole (MacLennan, 1933). In specimens fixed 
during division of the ciliate, the newly arising vacuolar regions some- 
times overlap the old ones and give the appearance of a division of the 
old one as described in Didinium (von Gelei, 1938), but a study of 
similar stages in living ciliates shows that they originate independently. 
These granules are continually migrating toward the vacuoles and dis- 
solving there, and no granules migrate outward, so the question of origin 
is shifted to the granules at the time they are scattered in the ectoplasm. 
No cases of division were observed, either in fixed or in living material, 
at any place nor at any stage of the life cycle. This negative proof is not 
entirely satisfactory, since these granules are small (0.25 — 0.50 [/.) and 
a very rapid division might escape notice. This problem does not occur 
in Ichthyophthirius, since all ectoplasmic granules (whether scattered or 
around the vacuoles) are absent in the encysted stage (MacLennan, 
1936), so that in this ciliate they must originate de novo in the young 
parasites, whether or not they continue from them by division as the 



148 CYTOPLASMIC INCLUSIONS 

ciliate grows during the feeding stages. Persistence and genetic continu- 
ity is restricted to a few very highly specialized types of vacuoles, and in 
most cases there is no continuity either in whole or in part. 

The osmiophilic reaction of the differentiated cytoplasm around the 
contractile vacuoles has given rise to statements that this is a lipoid 
structure (Nassonov, 1924; VolkonsI<y, 1933; Haye, 1930), and to the 
interpretation of the vacuolar system as due to the accumulation of 
lipoids at the vacuole-cytoplasm interface, which is similar to Parat's 
theory of the Golgi region around the vacuome. It is true that these 
structures are partially destroyed by lipoid solvents, but such evidence 
is not to be completely trusted and before acceptance must be corrobo- 
rated by more specific methods. In Ichthyophthirius these granules are 
negative to Sudan III and Nile blue sulphate. In Paramecium (unpub- 
lished work) I have used Ciaccio's long unmasking process and find only 
a very faint reaction — only slightly more than in the hyaloplasm — and 
negative results with Nile blue sulphate. These experiments show that 
there is no concentration of lipoids, either in the excretory granules or 
in the cytoplasm around the vacuoles. 

The variations in impregnation during the pulsatory cycle and during 
the life cycle suggest that the osmiophilic reaction is due to some reduc- 
ing agent (not a lipoid) which is poured into the vacuole during di- 
astole. The excretory theory of the contractile vacuole is indicated by the 
name nephridialplasm (Faure-Fremiet, 1925). The similarity between 
the cytological changes during the pulsatory cycle and in the glandular 
epithelium or renal tubules has been used in support of this theory (Nas- 
sanov, 1924, 1925). Further support was given by the demonstration 
that the osmiophilic granules dissolve in the vacuolar fluid which is then 
discharged (MacLennan, 1933). In Amoeba the activity of the contrac- 
tile vacuole is roughly proportional to the number of beta granules 
around it (Mast and Doyle, 1935b). It is certain in these cases that 
water plus some other material is being excreted. 

The question, then, is not whether excretion in a broad sense takes 
place, since water plus dissolved materials is certainly being excreted, 
but what are the dissolved substances which are carried to the vacuole in 
granular form? Nitrogenous excretion has often been assumed, but 
Weatherby (1927, 1929) found that the vacuolar fluid extracted by an 
application of the microdissection technique showed upon analysis too 












Figures 53-55. Excretory granules in living ciliates. Figure 53, from Polyplastron 
7nultivesiculatum; Figure 54, from Eudiplodiniiim maggii ; Figure 55, from Metadinium 
medium (from MacLennan, 1933). 



150 CYTOPLASMIC INCLUSIONS 

little nitrogenous materials — probably not more than one percent — to 
account for any significant part of the total excretion of Paramecium. The 
application of these results to all Protozoa is by no means certain, since 
I have shown previously (1933) that the pellicle of the Ophryoscolecidae 
is highly impermeable and the only pathway for the excretion of kata- 
bolic wastes is through the contractile vacuoles. Frisch (1938) has 
demonstrated a similar impermeability of the pellicle in Paramecium 
itself, and suggests that Weatherby's experiments be repeated, with the 
use of more delicate tests which have been devised recently. 

The materials concentrated in the osmiophilic structures of the con- 
tractile vacuole are not salts, since no significant accumulation is demon- 
strated in the nephridialplasm by microincineration (MacLennan and 
Murer, 1934). These materials may possibly be incidental in some 
species, but Amoeba dies if this elimination is prevented (Mast and 
Doyle, 1935b), demonstrating that these wastes are toxic and that in 
this respect they have the known properties of the nitrogenous wastes of 
metabolism. Evidence that these materials are the result of metabolism 
is found in Ichthyophthirius (MacLennan, 1936). The vacuoles are 
osmiophilic only during the active feeding phase, when large amounts of 
food are being used and converted into storage bodies; but during en- 
cystment, when these activities cease, the vacuoles are not osmiophilic. 
The food of this protozoan consists entirely of epithelial cells, which are 
largely proteins, but not more than a third of the reserves of the ciliate 
are proteins, the rest being carbohydrate and fat. During the feeding 
stage a large amount of the ingested protein would be deaminized to 
form other reserves, with the result that much larger amounts of nitroge- 
nous wastes would be formed during the feeding stage than in the en- 
cysted stage. 

Frisch (1938) suggests that the contractile vacuoles also function in 
respiration. This function cannot be correlated with the variations in 
the nephridialplasm in various Protozoa and thus, if this is a function of 
the contractile vacuoles, it is probably independent of the granules which 
are being considered here. 

Lipoid Reserves 

The lipoid materials considered in this section include all lipoids 
which are visible as granules and which are laid down during active 



CYTOPLASMIC INCLUSIONS 151 

feeding stages and are used during hunger or encystment. Zinger ( 1933) 
included all sudanophil particles as lipoid reserves, but digestive gran- 
ules, mitochondria, as well as other bodies respond to Sudan III because 
of their lipoid content. Zinger pointed this out, for in his conclusion he 
states that the sudanophil bodies are more than reserve materials. How- 
ever, until more is known of the functions of the intracellular lipoids, 
it is impossible to indicate accurately the boundary between reserve lipoids 
and those active directly in the metabolism of the cell. 

Lipoid reserves have been found in a large number of Protozoa (for 
a detailed list see von Brand, 1935). Usually, if not always, these gran- 
ules are in the endoplasm, either distributed at random, as in Ich- 
thyophthhius (MacLennan, 1936), or concentrated at one end, as in 
Anoplophrya (Eksemplarskaja, 1931). Although these visible lipoid 
granules occur in many Protozoa, they are not universal. Trypanosoma 
evansi lacks all lipoid reserves, a fact which is correlated with a lack of 
lipase (Krijgsman, 1936). The Ophryoscolecidae and Cycloposthiidae, 
noted for their tremendous glycogen reserves, have no important lipoid 
reserves. Mesnilella multispkulata has no lipoid reserves, although five 
other species of the same genus have many fat globules (Cheissin, 1930) . 

The formation of droplets of neutral fat inside a granule of fatty 
acid has been demonstrated in 0/?<^//Vzi^ (Kedrowsky, 1931) and Ich- 
thyophthirius (MacLennan, 1936) by the Nile blue sulphate method 
(Fig. 41). Since, after staining with Nile blue sulphate, very small 
quantities of fatty acid dissolved in neutral fat result in an intense blue 
color rather than the pink which is characteristic of pure neutral fats, 
the pink stain observed in the cases above indicates that there are no free 
fatty acids in the neutral fat granules, as would be expected if the fats 
were synthesized on the surface of these granules. The fatty acids and 
glycerine dissolved in the endoplasm are first segregated into granules, 
and in these granules the neutral fat is synthesized. Then this fat is segre- 
gated into the visible droplets of pure neutral fat inside the active gran- 
ules. These latter granules are typical Golgi bodies (MacLennan, 1936, 
1940), as indicated by the name endoplasmic Golgi bodies. However, 
since these are functionally an intermediate stage in the development of 
the fat reserves, the descriptive term "intermediate lipoid body" is more 
appropriate in a functional classification. 

In A. proteus the fat droplets grow in the cytoplasm without any in- 



152 CYTOPLASMIC INCLUSIONS 

termediate granules being visible. These cytoplasmic fat droplets are not 
derived directly from fat in the food vacuoles, but the ingested fat is 
absorbed as free fatty acid and glycerine, synthesized into neutral fat in 
the cytoplasm and then stored as granules (Mast, 1938) . Free fatty acids 
were demonstrated in the food vacuoles when fat was being digested, 
but none were demonstrated in the cytoplasmic fat droplets or on their 
surface when they were being formed. In the cases in which this process 
is not visible, either the synthesis is carried on elsewhere and the fat trans- 
ported to the granules as such, or the fatty acids are never allowed to 
accumulate sufficiently to show under the microscope. 

The visible lipoids, i.e., those which are found in definite globules 
and demonstrable by the ordinary fat-staining technique, include only 
a part of the total lipoids of the cell. "The pathologists have known for 
many years that the fats and fat-like substances of protoplasm are so 
bound or united to proteins as to be for the most part non-recognizable 
in the living or stained cell" (Heilbrunn, 1936). Besides the factors of 
food and the formation of fat from other substances such as carbohy- 
drate or protein, changes from bound lipoids to free globules must be 
considered in any estimation of the reserve lipoids. Heilbrunn demon- 
strated an increase in lipoid globules in specimens of A. proteus kept in 
a dilute solution of ammonium «salts. Three types of amoebae were found 
— those which show lipoids in culture, those in which lipoid globules 
appear after treatment with NH^Cl, and those in which no free lipoid 
appears even after treatment. In similar experiments with Arbacia eggs, 
Heilbrunn showed that the total lipoids of the protoplasm remained con- 
stant; therefore the newly visible bodies are derived from bound lipoids, 
not from new fat formation. Since ammonium salts in the culture medium 
raise the pW of the immersed cells, the results were attributed to alka- 
linization of the protoplasm. The fact that COo bubbled in the medium 
(which would tend to lower the protoplasmic /'H), inhibits the forma- 
tion of visible lipoids, confirms this hypothesis. Old cultures of Para- 
7necium show larger amounts of fat than new cultures, although the 
paramecia divide and show no ill effects (Zinger, 1933) ; and since such 
cultures contain ammonia (Weatherby, 1927), the presence of abnormal 
amounts of visible fats may be due to the resulting alkalinization of 
the protoplasm. 

Ultra-violet radiation causes a release of lipoid in Amoeba (Heil- 



CYTOPLASMIC INCLUSIONS 153 

brunn and Daugherty, 1938). The release of lipoids was greatly in- 
creased by a preliminary immersion in ammonium chloride solutions. 
"Further study is necessary in order to determine whether this fat re- 
lease is due to a direct action of the radiation on the protein-lipoid bind- 
ing or whether it may not be due indirectly to an alkalinization of the 
protoplasm" (Heilbrunn and Daugherty, 1938). In the same publica- 
tion it is stated that any stimulus in which localized increases in tempera- 
ture occur is efficient in the release of fat. This is also shown by the 
experiments of Sassuchin (1924), who compared the protoplasm of 
Opalina kept at room temperature with the protoplasm of those kept at 
35-38° C. In the first group he found elongate mitochondria in the endo- 
plasm (Kedrowsky's endosomes), but in the heated group only fat 
spherules and protein spherules, and these results were interpreted as due 
to the separation of mitochondria into their two components. These latter 
experiments should be repeated in individuals with little or no fat, and 
in species as to which there is more agreement on the identification 
of mitochondria. 

In ParameciujH (Zweibaum, 1921) 2ind Stentor (Zhinkin, 1930) fat 
is stored under conditions of low oxygen tension and lost when the oxy- 
gen tension is restored. The rate of loss in this case is dependent upon 
the temperature. 

Pathological conditions are often marked by fatty degeneration in the 
Protozoa. Degenerating coccidial oocysts show an increase in fat globules 
(Thelohan, 1894), and in Aulacantha fatty vesicles are formed and the 
nucleus is finally replaced by fatty bodies (Borgert, 1909). Individuals 
of Actinophrys which show depression by a lowered division rate and 
otherwise, have an abnormal number of lipoid bodies, and' in extreme 
cases show typical fatty degeneration. In the macronucleus of Payamec'mm 
parasitism by bacteria also results in tremendous quantities of visible 
lipoids in the cytoplasm and also of crystals (Fiveiskaja, 1929). 

Carbohydrate Reserves 

Granules containing carbohydrates are found in most Protozoa, al- 
though in a few species this reserve is in a diffuse form which is pre- 
cipitated as granules or irregular masses by fixation. The lack of any 
carbohydrate reserve at all has been proved in only a few species, such as 
Trypanosoma evans'i. 



154 CYTOPLASMIC INCLUSIONS 

The carbohydrate reserves in Paraniec'iuni ( Rammelmeyer, 1925) and 
in the cysts of Burs aria (Poljansky, 1934) are probably dissolved in the 
protoplasm, since they are visible in fixed specimens only as cloudy 
masses, not in regular granules. Homogeneous vacuoles, granules, or 
platelets visible in the living normal Protozoa are very common. They 
are well known in lodamoeha and other intestinal amoebae. Large num- 
bers of these granules are found in the flagellates from termites and 
wood-eating roaches (Cutler, 1921; Kirby, 1932; Cleveland, 1934; 
Yamasaki, 1937a). The carbohydrate granules of Stentor tend to be 
localized in a peripheral sheath of the endoplasm (Zhinkin, 1930) and 
just beneath the pellicle. In Arcella these granules are embedded in the 
chromidial net. In Ichthyophthirius these smaller granules are always 
associated with mitochondria (MacLennan, 1936). Glycogen granules 
are often associated with the parabasal bodies in flagellates (Duboscq 
and Grasse, 1933). 

Carbohydrate granules with definite internal structure are by no means 
uncommon. The granules of Sporozoa (Fig. 60) have a cross or star- 
shaped center (Joyet-Lavergne, 1926a; Daniels, 1938), the general ap- 
pearance of which and ability to accumulate iodine suggest vacuoles. 
Identification of a lipoid center (Erdmann, 1917) is based on insufiicient 
evidence and, in view of the later work quoted above, seems unlikely. 
Vacuolated bodies are also found in Balantidium (Fig. 57) with the 
added feature of crystals floating in some of the vacuoles (Jirovec, 
1926) . Two types of granules are found in Dif^ugia, small homogeneous 
spherules and larger elliptical bodies with a center granule which stains 
a pale blue after hematoxylin and a rim which is rose-colored after Best's 
stain (Rumjantzew, 1922). 

In the Ophryoscolecidae, the granules possess a spherical center (Fig. 
56) denser than the rest of the granule (MacLennan, 1934). The most 
spectacular of the carbohydrate reserves are the skeletal plates of the 
Cycloposthiidae, Ophryoscolecidae, and related families. The plates 
themselves are probably supporting structures, but in their meshes are 
platelets of the same type as the scattered cytoplasmic granules. The plate- 
lets in the Cycloposthiidae (Fig. 61) are roughly spool-shaped with slen- 
der strands connecting the flanges of adjacent granules (Strelkow, 
1931), but in the Ophryoscolecidae (Fig. 62) the polygonal plates are 
unconnected (MacLennan, 1934). 



CYTOPLASMIC INCLUSIONS 155 

The formation of paraglycogen bodies has been followed in only a 
few cases. The bodies of Peloniyxa behave like permanent bodies with a 
protein stroma and with the paraglycogen being built up or released as 
the case demands (Leiner, 1924). The paraglycogen granules of Poly- 
plastron are likewise independent of other formed components and are 
apparently self-perpetuating (Fig. 56), since they show regular division 
(MacLennan, 1934). The dense centers may be naked or, more often, 
surrounded by an envelope of varying thickness. In the largest of these 
compound granules, the centers are dumb-bell-shaped or double, and 
in the latter the envelope also is constricted. These stages probably repre- 
sent growth or utilization stages and division stages, although this was 
not confirmed by following a single granule in live ciliates. The com- 
plex granules of Amoeba hydroxena (Fig. 58, 59), in which a varying 
number of glycogen granules are imbedded in glycoproteid (Wermel, 
1925), suggests a conversion of glycogen into glycoprotein for storage 
and the reversal of this process in utilization. 

Some paraglycogen granules are formed in association with mito- 
chondria instead of being independent bodies. The paraglycogen in 
Ichthyophthirius first appears as a minute vacuole (Fig. 17) in the center 
of a sphere of mitochondrial material (MacLennan, 1936) . As the gran- 
ules grow, this mitochondrial shell breaks into short rods fused to the 
surface of the paraglycogen. The mitochondria disappear after the gran- 
ule has attained full size. Joyet-Lavergne ( 1926b) also noticed a morpho- 
logical relationship between mitochondria and paraglycogen of greg- 
arines, but says "il y a la un simple rapport de contact et nous n'avons 
aucune raison de suppose une intervention dans da genese du para- 
glycogene." However, in the case of a granule in the center of an un- 
broken sphere, as in Ichthyophthirius, it is difficult to list the relationship 
as merely an incidental contact. 

Duboscq and Grasse (1933) show that the glycogen granules of 
Cryptobia helicis are not found scattered in the cytoplasm, but are formed 
in close contact with or in the strands of the parabasal bodies (Fig. 65 ) . 
The glycogen is not laid down in the summer, but only in the winter, a 
fact which, they point out, would explain the negative results of other 
authors. This formation of glycogen by the parabasal body parallels the 
secretion of protein granules by the macronucleus of ciliates — a part of 
the segregation function which, in most species, is performed by isolated 




56 






57 





59 





mm 

& 

Figures 56-64. Carbohydrate reserves. Figure 56, stages in the paraglycogen granules 
of Polyplastron multivesiculatum, Champy-osmic impregnation followed by Sudan III in 
hot paraffin (after MacLennan, 1934) ; Figure 57, growth of crystals in paraglycogen 
granules of Balantidium elongatum, Zenkers-dahlia (after Jirovec, 1926) ; Figures 58- 
59, glycogen droplets in a glycoproteid granule, from Atnoeba hydroxena, Carnoy-Best 
(after Wermel, 1925); Figure 60, vacuolated paraglycogen body from Sporozoa (after 
Joyet-Lavergne, 1926) ; Figure 6l, skeletal platelets of Cycloposthium edentatum, Lugol 
(after Strelkow, 1929) ; Figure 62, skeletal platelets of Polyplastron fnultivesiculatum, 
Champy-osmic-Sudan III in hot paraffin (after MacLennan, 1934) ; Figures 63-64, 
glycogen reserves in Trichonympha ag'ilis, Best's stain, 63 normal, 64 showing loss of 
glycogen just before death under conditions of lowered temperature and raised oxygen 
pressure (after Yamasaki, 1937). 



CYTOPLASMIC INCLUSIONS 157 

cytoplasmic granules, is performed in the one case by a nuclear structure 
and in the other case by a neuromotor structure. 

The differentiation between the various carbohydrates found in the 
Protozoa is based on their staining reactions and solubility, since the 
exact nature of the sugars involved in the formation of protozoan poly- 
saccharides is unknown. Zhinkin (1930) and von Brand (1935) pointed 
out that this is unsatisfactory and contend that no separation should be 
made from glycogen until this is known. However, the differences are so 
pronounced that it is convenient to retain the name paraglycogen. 

Soluble glycogen as found in vertebrate liver cells is relatively rare. 
The diffuse materials found in Paramecium and Bursaria are probably 
of this type. The commonest carbohydrate is paraglycogen, distinguished 
by Biitschli (1885) from glycogen on the basis of its relative insolubility 
in water as compared with true glycogen. It is digested by ptyalin and 
diastase and the sugar produced reduces Fehling's solution. It stains a 
light brown in iodine and brown or brown purple in iodine-sulphuric 
acid or chlor-zinc-iodide. Probably all of the granular reserves of carbo- 
hydrate in Protozoa are paraglycogen or some similar relatively insoluble 
compound. The reserve granules of the flagellates of termites have been 
identified as glycogen (Yamasaki, 1937a; Kirby, 1932); but in the re- 
lated flagellates of the wood roach, since the Protozoa contain no enzyme 
capable of breaking down glycogen, it has been suggested that the gran- 
ules which stain with iodine consist of some other product which results 
from the breakdown of cellulose (Cleveland, 1934). The material in 
the platelets of the Ophryoscolecidae has been named ophryoscolecin on 
the ground that it is unique in this family and is more like cellulose than 
paraglycogen (Dogiel and Fedorowa, 1925). It was later identified as a 
hemicellulose (Strelkow, 1929). This interpretation is based on slight 
variations in solubility and color reactions, but other authors, using some 
of the same methods and some different methods, were not able to find 
any difference between the reactions of paraglycogen and the platelets 
(Schulze, 1922, 1924, 1927; Weineck, 1931, 1934; MacLennan, 1934). 
However, such arguments cannot be settled, as von Brand suggests, until 
the exact structure of these polysaccharides is known, and the term para- 
glycogen in this discussion is used in a rather general sense for carbo- 
hydrates more insoluble in water than glycogen and differing in color 
reactions from starch and cellulose. 



158 



CYTOPLASMIC INCLUSIONS 



The presence of more than one type of material in the same granule 
has been demonstrated in several cases, in spite of the relative crudity 
of the cytochemical methods for the demonstration of carbohydrates. The 
oval carbohydrate bodies of Difjlugia are not completely dissolved in 
ptyalin, and their staining reactions suggest the presence of a glyco- 
proteid (Rumjantzew, 1922). Two types of carbohydrate reserves have 
been reported from Sporozoa by Dobell (1925). Chakravarty (1936) 




Figure 65. The associa- 
tion between glycogen and 
the parabasal body in 
Cryptobia helicis, winter 
forms stained with iodine. 
(After Duboscq and 
Grasse, 1933.) 



also differentiated two sets of granules by differences in the speed of 
destaining after treatment in iodine. These authors refer to one set as 
glycogen, the other as paraglycogen. Two types of carbohydrate have 
been found in Actinosphaerium (Rumjantzew and Wermel, 1925), and 
were identified as glycogen and a glycoprotein on the basis of their re- 
action to Best's and Fischer's stains. Peloniyxa loses its paraglycogen dur- 
ing prolonged starvation, but since the remnants of these granules may 
be stained with haematoxylin (Leiner, 1924), it is probable that the 
carbohydrate is here associated with a protein. The carbohydrate bodies 



CYTOPLASMIC INCLUSIONS 159 

of Amoeba hydroxena contain two different materials (Figs. 58, 59) 
and on the basis of Best's and Fischer's methods have been interpreted 
as granules of glycogen embedded in glycoprotein. 

The decrease in glycogen or paraglycogen during hunger or encyst- 
ment, and its storage during the feeding stages, has been noted so often 
that detailed descriptions of observations under controlled conditions 
are rare. The glycogen in Stentor is deposited during low temperatures 
and utilized at higher temperatures, and this process is accelerated by 
starvation (Zhinkin, 1930). Fat, rather than glycogen, is deposited, if 
the oxygen tension is lowered. Under such conditions some of the car- 
bohydrate is probably converted into fat. In Trkhonympha also, avail- 
able food, oxygen tension, and temperature affects the amount of glyco- 
gen present ( Yamasaki, 1937b) . The cytoplasm of this species is divided 
into two parts by a fibrillar basket, which suspends the nucleus from the 
anterior cone of the body and separates this portion from the rounded 
posterior part in which the food vacuoles are formed. Both regions 
normally contain glycogen (Fig. 63), but during starvation the glycogen 
in the posterior part disappears first, the glycogen in the anterior part 
then diminishes and disappears, and the death of the organism follows 
shortly. Similar results are observed at high temperatures, or with oxy- 
genation at room temperature. However, when the termites are oxygen- 
ated at low temperatures (Fig. 64), the glycogen in the posterior por- 
tion often shows little change, but the portion anterior to the nucleus 
disappears rapidly. As soon as the glycogen in the corbula disappears the 
protozoan dies, even though glycogen remains in the body region. 
Yamasaki states that the posterior region is simply one of synthesis and 
storage, while the anterior region is the region of consumption. He 
concludes that defaunation by oxygen is due not only to, toxicity but also 
to a depletion of the glycogen available for the nucleus and motor or- 
ganelles. 

Trypanosoma evansi possesses no glycogen and, since it possesses no 
amylase, is not able to synthesize it (Krijgsman, 1936). Other trypano- 
somes do deposit glycogen, but at best it forms an insignificant reserve, 
since trypanosomes may use three times their body weight in sugar in 
twenty-four hours (von Brand, 1938). In this case the glycogen re- 
serves of the trypanosomes are the liver glycogen of the host. 



160 CYTOPLASMIC INCLUSIONS 

Protein Reserves 
This term is one of convenience and, as in the case of the term Hpoid, 
cannot be taken in a strict sense, but is used here to include, besides 
true proteins, bodies which contain hpoids or carbohydrates, as well as 
proteins, amino acids, nucleic acid, and so forth. Since most of the fixing 
agents precipitate at least the protein portions of such granules, many 
have been described, although relatively few have been identified by 
acceptable microchemical methods. For this reason they have been de- 
scribed under a variety of names, many of which mention incidental 
staining properties. Some of the names which are most securely em- 
bedded in the literature are chromidia, volutin, metachromatic granules, 
basophilic granules, chromatoidal bodies, and albuminoid reserves. The 
confusion in these terms is best illustrated by chromidia. This was orig- 
inally used to designate chromatin bodies which are extruded into the 
cytoplasm from the nucleus (Hertwig, 1902) and which have the abil- 
ity to reaggregate to form new nuclei. Although this interpretation has 
been disproved, the name may be retained to designate these granules 
(Meyers, 1935). In other cases it is used to designate nonchromatin ma- 
terial which is supposed to be extruded from the nucleus (Dapiels, 
1938). Other authors use it even more loosely to designate basophilic 
and metachromatic cytoplasmic bodies which are secretory in nature 
(Campbell, 1926). The elimination of the original meaning was due to 
the improvement of both cytoplasmic and nuclear methods, accompanied 
by detailed studies of life cycles. The last stronghold of this theory — 
the Foraminifera — was eliminated by the tracing of the nuclear history 
in live Patellina throughout the vegetative and sexual stages, with a 
complete demonstration of the cycle with moving pictures (Meyers, 
1935). The exclusion of chromidia in the original sense, with respect 
to the cells of the Metazoa, has already been accepted (Wilson, 1928). 
Many chromidia are actually mitochondria ( Faure-Fremiet, 1910) 
which contain a high percentage of protein and are therefore resistant 
to routine fixatives. This probably led to one revival of the chromidial 
theory, according to which all cytoplasmic structures are formed from 
mitochondria, which in turn originate from the nucleus as chromidia. 

Alexeieff in a series of works on the Flagellata strives to prove that all cell 
structures are formed at the expense of mitochondria. The latter, according to 
Alexeieff, in their turn are not autonomic, as the majority of investigators 



CYTOPLASMIC INCLUSIONS 16 1 

suppose, but originate from the nucleus as chromidia. ... In cases where 
the autonomy of the mitochondria and of the blepharoplast is indisputable, 
this author always attributes to them a nuclear origin though phylogenetic. 

After this summary of Alexeieff's theory, Milovidov (1932) rejects it. 
Certain cytoplasmic granules of Uroleptus are derived from the nuclei 
during reorganization (Calkins, 1930), and these granules were de- 
scribed as mitochondria, but since they do not stain with janus green 
they do not seem to be typical mitochondria. 

The term chromidia, as now accepted, includes cytoplasmic granules 
supposed to be derived from the nucleus (but not necessarily chromatin) , 
particularly in the Sporozoa. It also includes granules in the rhizopods, 
at one time supposed to be examples of the chromidial theory, but now 
retained without any such implication. 

The chromidial net, characteristic of many of the rhizopods with shells, 
is a definite morphological entity which may be recognized independently 
of particular staining methods. The net itself is negative to Feulgen's 
stain in Arcella and Chlamydophrys, either with or without hydrolysis, 
and is digested more rapidly than the nucleus by pepsin or trypsin 
(Reichenow, 1928). Since the net in Dijjlugia gives a positive reaction 
with Ciaccio's lipoid method (Rumjantzew, 1922), it probably has a 
lipoid component in addition to the protein component in this species. 
Although basophilic, it is not directly related to the nuclear material. 
On the other hand, the net of Pdtell'ina is positive to Feulgen's method, 
but complete studies show that it is independent of the nuclei (Meyers, 
1935). In both cases the net is a specialized mass of reserve protein, and 
within it may be found two other types of reserve, volutin and glycogen 
granules. This is not true in all species, since no glycogen is found in the 
net of Difjlugia (Rumjantzew, 1922). The chromidia of gregarines are 
similar in ordinary staining reactions to the karyosome and to the pro- 
tein reserves (Daniels, 1938). 

The chromidia of several Sporozoa (Joyet-Lavergne, 1926a) are posi- 
tive to Millon's reagent and are therefore certainly protein and they 
appear to be associated with mitochondria. In gregarines from meal- 
worms, on the other hand, these granules are negative to both Millon's 
reagent and Feulgen's reagent, and show no morphological relationship 
with mitochondria (Daniels, 1938). Daniels found chromidia and volu- 
tin similar in shape, distribution, and so forth, but found fewer black 



162 CYTOPLASMIC INCLUSIONS 

granules in haematoxylin preparations than blue granules after the 
methylene blue method. She concludes that they are separate types of 
granules, although there is a close relationship. Joyet-Lavergne was not 
able to decide whether chromidia and volutin are really separate. 

Daniels observed buds on the surface of the karyosome, then bodies 
in the nuclear sap, and finally in the cytoplasm near the nucleus, but she 
found no direct evidence as to how they penetrate the nuclear mem- 
brane. On the basis of these suggestive observations, she concludes that 
these bodies are derived from the karyosome. With regard to the validity 
of this conclusion, the comment of Wilson (1928, p. 96) with regard 
to a similar case in oogenesis is highly pertinent: "To the writer none of 
these cases yet seems to be satisfactorily demonstrated, and the question 
is a most difficult one to be settled by studies on fixed material alone." 
Joyet-Lavergne ( 1926a) calls these protein granules albuminoid reserves, 
a name far more appropriate than chromidia, which at least implies a 
nuclear origin. 

Volutin granules are basophilic granules which are also metachromat- 
ic. Because of their pronounced basophilia, volutin granules have often 
been linked with chromatin. However, they are negative to Feulgen's 
stain after hydrolysis, but give a positive reaction when the preliminary 
hydrolysis is omitted (Reichenow, 1928), a characteristic of free nu- 
cleic acid. The full Feulgen reaction apparently dissolves this type of 
volutin granule, so that in Arcella there results a diffuse Feulgen reac- 
tion in the chromidial net. This is a possible explanation of the positive 
Feulgen test by the chromidial net of Patell'/na. The volutin granules of 
Trypanosoma melophagium contain no nucleic acid (van Thiel, 1925), 
while those of T. equinmii do (Reichenow, 1928). The volutin bodies 
of r. evansi were not tested in this respect (Krijgsman, 1936), although 
they are listed as containing nucleic acid. Since reserve bodies are not 
the same in all species of the genus (some trypanosomes are able to 
store glycogen while others are not, according to von Brand, 1938), 
both analyses of the basophilic granules may be correct. 

Volutin granules increase and nuclear granules decrease in trypano- 
somes which have been treated with atoxyl (Swellengrebel, 1908). This 
fact in conjunction with the staining reactions of volutin, were inter- 
preted as indicating a direct nuclear origin — in other words, a type of 
chromidia. In these experiments the results are probably a degeneration 



CYTOPLASMIC INCLUSIONS 163 

phenomenon, since in Velomyxa the expulsion of chromatin into the 
cytoplasm is found just prior to death (Schirch, 1914). Hindle (1910) 
thought of this as a degeneration phenomenon in T. gamhiense. In 
various phytoflagellates division stops when volutin is lost, and the volu- 
tin was interpreted as a nuclear reserve (Reichenow, 1928), although no 
direct connection between the two was demonstrated. A dehydrase has 
been demonstrated in Trypanosoma by the leucomethylene blue method 
and localized in the volutin granules (Krijgsman, 1936). Krijgsman, 
however, holds to Reichenow's views of volutin as a nuclear reserve. 

Protein bodies in Oxymonas dinwrpha. which are negative to Feul- 
gen's stain and stain with either basic or acid dyes (i.e., not metachromat- 
ic), have been called volutin granules (Connell, 1930), although they 
are not volutin in the sense used by Reichenow. However, in Oxymonas, 
as in the phytoflagellates of Reichenow's experiments, division ceases 
when these granules are exhausted. Since the division stages of Oxymonas 
are also the flagellated stage, the protein granules could be explained 
as reserve bodies for the expenditure of energy by these organelles. 
Neither explanation has adequate proof, since each merely correlates 
obvious phenomena. 

Volutin is thus a term which has no standard usage, but wherever 
microchemical tests have been made volutin has been found to contain 
proteins, nucleic acid, or other similar materials. Since the available evi- 
dence shows that it behaves as a reserve material, it seems to me to be 
convenient to include it as one of the various types of protein reserves 
and to eliminate the terms volutin and metachromatin, neither of which 
seems to have been used consistently by protozoologists. 

The macronuclei of many ciliates contain one or more large, intensely 
basophilic bodies lodged in vacuoles among the closely packed granules of 
chromatin ( Chakravarty, 1936; MacLennan, 1936). Since their num- 
ber and size vary, it has been suggested that these are reserve materials 
(Kazancev, 1928). In Ichthyophthirius these granules have been traced 
in living ciliates from the macronucleus through temporary breaks in the 
macronuclear membrane into the cytoplasm (Fig. dd) where they are 
stored until resorption and utilization occurs in the encysted stages (Mac- 
Lennan, 1936). These bodies take both acid and basic dyes even more 
strongly than chromatin and, unlike the chromatin, are negative to 
Feulgen's reaction and Macallum's tests for iron. These granules first 



164 



CYTOPLASMIC INCLUSIONS 



appear as minute bodies at the lower limits of visibility, embedded in 
the chromatin net during the feeding stages of the ciliate but not during 
encystment. The macronucleus is positive to Feulgen's reagent without 
preliminary hydrolysis during the formation of these granules, but at 
no other time. It seems probable that food materials are built up in the 
macronucleus into chromatin, which is then split into a group contain- 
ing iron and nucleic acid and another protein group which lacks these 
substances. The first group is used to rebuild more chromatin and the 
latter group is segregated into the granules which are ejected into the 




Figure 66. The forma- 
tion and release of protein 
granules from the macro- 
nucleus of Ichthyophthi- 
ritis mullifiliis, Feulgen- 
light green. (After Mac- 
Lennan. 1936.) 



cytoplasm. Since granules of this type are found in both the macro- 
nucleus and the cytoplasm of so many ciliates, this is probably quite a 
general phenomenon. The Protociliata lack macronuclei, but perform 
this same function by the segregation apparatus. 

The balls of chromatin and other macronuclear fragments which are 
extruded during the various types of macronuclear reorganization are 
only incidentally reserve material, if at all, and will be considered in 
detail in the chapters on nuclear phenomena. 

The crystals which are common in various Protozoa are often con- 
sidered to be excretory products, and in some cases have been identified 
as uric acid (for a discussion of this work, see Reichenow, 1929) . Recent 
work (Mast and Doyle, 1935b) shows, however, that some of the crystals 
must be regarded as reserve material. A. proteus contains two types of 
crystals, a bipyramidal type and a plate-like type, which are suspended 
in vacuoles containing an alkaline fluid. A careful study of spectroscopic 



CYTOPLASMIC INCLUSIONS 165 

analysis, solubility, and form shows that the bipyramidal type probably 
consists of a magnesium salt of a substituted glycine. The plate-like 
crystals are insoluble in a saturated solution of leucine, and in structure 
resemble leucine crystals. If the crystals are removed by centrifuging 
and the Amoeba is then put in a solution which contains amino acids 
and egg albumin, the platelets are formed in the vacuoles which con- 
tain leucine, while the bipyramidal crystals are formed in all solutions. 
"Crystals are normally formed from amino acids derived from food dur- 
ing digestion" (Mast and Doyle, 1935b). These crystals decrease in 
number just before the refractive bodies increase in number, indicating 
that the crystals are an intermediate stage in the transfer of food from 
the food vacuoles to the lipoprotein refractive bodies. 

The chromatoidal bodies of various parasitic amoebae, are intensely 
basophilic structures, the fixing and staining reactions of which suggest 
a protein composition. They possess neither chromatin nor free nucleic 
acid (Reichenow, 1928), so are not volutin; but they are similar in 
their reactions to the protein bodies of ciliates. Since they disappear dur- 
ing encystment they are reserve bodies. 

The reserve proteins are often found in combination with other ma- 
terials. In Amoeba, lipoids and proteins are bound together in the re- 
fractive bodies (Mast and Doyle, 1935a). In Act'mosphaerium, granules 
of glycoproteid are present (Rumjantzew and Wermel, 1925). Similar 
inclusions are found in Ophryoglena (Zinger, 1928). In the Foet- 
tingeriidae the protein reserves have the characteristics of the vitellin of 
the hen's Qgg and in Polysp/ra there is a single central mass consisting of 
protein associated with a carotenoid (Chatton, Parat, and Lwoff, 1927). 
The protein portion alone is used, the carotenoid remaining in the old 
cyst, and finally disappearing during encystment. 

The protein bodies which are found throughout the Protozoa vary 
greatly in their specific structure and composition. This variation, with 
the resulting variation in staining reactions, has resulted in a complicated 
nomenclature with the usage of terms proposed by each author. The 
term chromidia is so definitely bound up with disproved theories that 
it should be dropped. Volutin should either be dropped or definitely 
restricted to metachromatic granules which respond to Feulgen's stain 
when used without hydrolysis. Whenever the function is that of a re- 
serve, as in the majority of known cases, I believe these granules should 



166 CYTOPLASMIC INCLUSIONS 

be called simply protein reserve bodies, a usage found convenient by 
Joyet-Lavergne (1926a). At the same time, it should be recognized that 
a reserve function is the one most easily identified by morphological 
methods, and that other functions must be investigated. The presence 
of dehydrogenase (Krijgsman, 1936) in protein granules of Trypano- 
soma evansi is one definite lead. 

The protein reserve bodies are as catholic in origin as in structure and 
may result from the activities of the segregation granules, macronucleus, 
mitochondria, or food vacuoles, or may be independent of other formed 
bodies. The crudity of our knowledge of cytoplasmic granules is illus- 
trated by the fact that no suggestion of the significance of these differ- 
ences can be made. 

External Secretion 

This important cytological subject has been greatly neglected in the 
Protozoa and is generally ignored in a discussion of the protozoan cyto- 
plasm, except as the vacuolar apparatus is considered to be a secretory or- 
ganelle. It is an important subject in itself and is most nearly comparable 
to secretion studied in the Metazoa. 

The attaching organs, or at least the cementing portion, of sessile 
Protozoa are secreted structures. Just preceding the formation of the 
peduncle of Campanella, granules are found in the basal region (Faure- 
Fremiet, 1905 ) . Tintinnopsis nucula is cemented to the lorica by a mucus 
secretion which is derived from basophilic granules in the stalk (Camp- 
bell, 1926). 

The lorica of Favella is likewise derived from cytoplasmic granules 
(Campbell, 1927). Granules which are to form the new lorica accumu- 
late near the mouth, in dividing animals. After division these are forced 
out through the cytostome, expand, fuse, and harden. At the same time 
fecal pellets are molded into this secreted material and the whole lorica 
is shaped by the activities of the motor organelles. There is in this form 
a local zone of secretion, as in gland cells, not a general secretion over 
the whole surface. 

The shell of Eaglypha is formed from separate shell plates, which are 
secreted within the cytoplasm (Hall and Loefer, 1930). They appear 
first as small refractive spheres in vacuoles, then enlarge and elongate to 
become typical shell plates. (Fig. 67). The finished plates lie free in the 



CYTOPLASMIC INCLUSIONS 



167 



cytoplasm. It was demonstrated that the reserve plates have no connection 
with either the mitochondria or the neutral red globules. 

The cyst of Ichthyophthirius is secreted in two parts: first a homo- 
geneous clear membrane is formed (Fig. 68) and then individual 
fibrils are extruded, apparently between the bases of the cilia (Mac- 
Lennan, 1937). These sticky fibrils are stroked into rope-like fibers, 
which adhere to the under side of the outer membrane (Fig. 69) by 
the activities of the cilia. Although seven types of granules were demon- 



O © «) (») ill 



67 



wmrmmm 



66 



69 



Figures 67-69. External secretion. Figure 67, inclusions in Euglypha alveolata, prob- 
ably representing the formation of reserve shell plates (after Hall and Loefer, 1930) ; 
Figures 68-69, secretion of the cyst wall in Ichthyophthirius multifiliis ; Figure 68, 
section of early stage showing only the homogeneous layer; Figure 69, section of 
later stage showing the addition of the fibrillar layer (from MacLennan, 1936). 

strated, no granules could be associated with the secretion of this cyst. 
In Nyctotherus, variations in the thickness of the secreted cyst may be 
correlated with the distribution of ectoplasmic structures (Rosenberg, 
1937), but no granules responsible for the secretion were noted in this 
form either. The lorica of Folliculiria ampulla is likewise secreted in the 
form of a clear fluid, which hardens to form a membrane just beyond 
the tips of the somatic cilia ( Faure-Fremiet, 1932). 

The secretion of vacuoles of oxygen in Arcella is not associated with 
granules, but with nongranular regions of hyaloplasm, and is probably 
a result of oxidative and reductive processes of the cell (Bles, 1929). 

The most striking aspect of these examples of protozoan secretion is 



168 CYTOPLASMIC INCLUSIONS 

that none of them has been traced to Golgi bodies, mitochondria, or 
segregation bodies — i.e., they do not react with osmic acid, Janus green 
B, nor neutral red. It is evident that not all important segregation nor 
synthesis is revealed by these stains. 

The mucus granules of Euglena (Dangeard, 1928) and the pellicular 
secretions of Vortkella (Finley, 1934) stain with neutral red and thus 
might be classed the segregation granules of Opalina and others, the 
only difference being that in the former is segregated mucus, which is 
not used within the cell but is extruded in the normal functioning of 
the protozoon, while in the latter are segregated proteins, which are 
normally used within the cell. It is interesting, however, that Kedrowsky 
found that when the segregation granules were filled with foreign ma- 
terials, such as the organic silver compounds, the granules are extruded. 
The expulsion of droplets containing neutral red may be induced in 
Paratnecium (Frisch, 1938) and other ciliates. These examples indicate 
that the formation of the segregation granules and the secretion granules 
is comparable, the only difference being that in one the material is used 
internally and in the other externally. 

The Granular Complex 

The detailed consideration of each of the types of cytoplasmic gran- 
ules has resulted in the conclusion that there are no universal cyto- 
plasmic components and that each of the terms mitochondria, Golgi 
bodies, neutral red granules, and so forth has been applied to a hetero- 
geneous assortment of granules of widely different functions. This con- 
clusion, derived from a consideration of the types of granules separately, 
becomes inescapable if we consider the whole granular complex. The 
problem is on the surface one of classification, but fundam.entally it is 
one of function — what functions are performed by cytoplasmic gran- 
ules, and is the same function always performed by the same type of 
granule in different Protozoa.'' Since these granules are not independent 
units but are part of a granular complex which in turn is a part of 
the whole cell, this whole complex must be considered in seeking an 
answer to these problems of function. The investigations which seem to 
be suitable for this comparison are those of Mast and Doyle (1935a, 
1935b), Holter and Kopac (1937), Holter and Doyle (1938), all on 
Amoeba proteus; Hopkins (1938a, 1938b) on Flahellula mira; Ked- 



CYTOPLASMIC INCLUSIONS 169 

rowsky (1931-33) on OpaUna ranarum; MacLennan (1936, 1937) on 
Ichthyophthirius multiflns; and Joyet-Lavergne (1926 on) on several 
Sporozoa, supplemented by the observations and experiments of Daniels 
(1938) on similar species. Since a summary of the individual granules 
has been given in the previous sections, only a general account of each 
granule will be given in this comparison. 

The number of types of granules ranges from two in the marine 
amoeba Flabellula to at least six in Ichthyophthirius and some of the 
Sporozoa. In Flabellula there are only digestive granules and small gran- 
ules of unknown composition and function. This small number contrasts 
sharply with A. proteus, which has four types of granules of cytoplasmic 
origin: refractive bodies ( dictyosomes ) , alpha granules unknown in 
composition and function, mitochondria (beta granules), and neutral 
fat granules. In addition to these granules there are two types of crystals, 
blebs on these crystals, and vacuole refractive bodies, all of which arise 
in connection with the food vacuole. The two ciliates Opalina and Ich- 
thyophthirius also show marked differences in number of granules — the 
former with only four types and the latter with seven. Opalina has segre- 
gation bodies, endosomes (mitochondria?), intermediate lipoid bodies 
(endoplasmic Golgi), and neutral fat, while Ichthyophthirius has inter- 
mediate lipoid bodies (endoplasmic Golgi), neutral fat, excretory gran- 
ules (ectoplasmic Golgi), mitochondria, paraglycogen, and protein 
bodies. Gregarines and Coccidia have at least one type of Golgi body, 
neutral fat, one or two types of mitochondria, paraglycogen, one or two 
types of protein reserves, and neutral red bodies, a total of six to eight 
types of granules, allowing for differences in the accounts of Daniels and 
Joyet-Lavergne. Not all the Sporozoa present such a complicated picture, 
since there are probably not more than three types of granules in Plasmo- 
dium: mitochondria, segregation granules, and pigment. These marked 
differences, which appear even with a crude comparison based only on 
number of types of granules, show clearly that at best only very few gran- 
ules could be universal. Furthermore, the number of granules varies in- 
dependently of the relationships of the Protozoa involved, since both large 
and small numbers of granules are found in Protozoa of the same class. 
The immediate facts which stand out with respect to the staining re- 
actions of these five species of Protozoa are that in each species are 
granules or vacuoles which are stained specifically by Janus green B 



170 CYTOPLASMIC INCLUSIONS 

and other mitochondrial stains, in each are granules which segregate 
neutral red, and in each, with the exception of Flabelhda, are granules 
which may be specifically impregnated by the Golgi methods. If we do 
not press the comparison any further, we can say that chondriome and 
vacuome are universal cell constituents but, even with the very crude 
definition based on impregnation alone, Golgi bodies are lacking in one 
of the five Protozoa discussed. However, this apparent uniformity 
is reduced if these granules are compared with respect to other character- 
istics than the so-called specific staining methods, which are in reality 
quite crude in spite of brilliant contrasts. With respect to mitochondria, 
the bodies range from one extreme of temporary induced granules in 
Flabellula to two separate types in some of the Sporozoa. The neutral 
red bodies are even less comparable: in Ichthyophthirius they are lipoid- 
containing bodies, which are found only in association with gastrioles. 
In Opalina they range from watery vacuoles to dense bodies of proteins 
and are obviously not connected with any gastrioles. In Flabellula they 
are vacuoles in which is also dissolved the material stainable with Janus 
green. In Amoeba there are two types of neutral red bodies which are 
of cytoplasmic origin, and both contain large amounts of lipoids and at 
some stages proteins as well. One of these types, the refractive bodies, 
are apparently built up from material derived from the blebs and the 
crystals. In a structural sense also the refractive bodies are unique and 
are much more complex than any of the other neutral red bodies. In 
the Sporozoa, lipoid dictyosomes are weakly stainable with neutral red, 
but in addition there are non-lipoid bodies which stain much more 
specifically with neutral red. With respect to Golgi bodies, even if we 
ignore Flabellula, harmony is not achieved. In Amoeba the two types 
of neutral red bodies, as well as the mitochondria, respond to impregna- 
tion and bleach with difficulty. A comparison between these lipoid bodies 
and the endoplasmic Golgi bodies (intermediate lipoid bodies) of 
Ichthyophthirius, Opalina, and the dictyosomes of Sporozoa seems quite 
logical, until we consider that in these latter three species, these fatty 
acid bodies are an intermediate step in the formation of neutral fat, 
while the refractive bodies of Amoeba are finished bodies, the granules 
of neutral fat being morphologically independent of them. None of these 
lipoid bodies is comparable with the non-lipoidal ectoplasmic Golgi 



CYTOPLASMIC INCLUSIONS 171 

bodies (excretory Golgi) around the contractile vacuoles of Ichthy- 
ophthmus. 

These comparisons demonstrate that with respect to actual composition 
not even one single type of granule is found throughout these five species 
of Protozoa, and the apparent universal presence of certain types of 
granules is due to the lack of specificity in Janus green, neutral red, 
osmic acid, and other stains. This is demonstrated in spite of the fact 
that the known composition of these bodies can be stated only in 
qualitative terms which are actually very broad — i.e., lipoid, non-lipoid, 
protein, and so forth. 

A comparison on the basis of composition alone is open to criticism 
if it is not checked from other angles. The segregation apparatus of 
Opalina may be aqueous vacuoles, dense protein bodies, or it may contain 
bile pigments, depending on the medium and the temperature. Using 
composition alone as a criterion (or the neutral red reaction, for that 
matter), these granules would be separately classified, actually Ke- 
drowsky showed they are the same granules with the same function — 
segregation and synthesis. In this case the stain reactions and the compo- 
sition are incidental, and they are important in the classification of the 
segregation apparatus only if they can be used to reveal the function. 
Can groups of granules be demonstrated if the granules are compared, 
not on the basis of structure nor of composition, but on function? If so, 
are any of these groups represented in all of these five species which 
we are considering? This comparison cannot be in any way as complete as 
the comparison based on staining reactions and composition, since for 
the most part this knowledge is restricted to those functions which have 
a definite morphological expression — digestion, storage, external secre- 
tion, and so forth. The apparent emphasis on these functions should not 
be considered as an implication that these are the only functions in which 
the cytoplasmic granules may play a role, but as an inverse expression 
of the difficulties of localizing functions which do not produce visible 
structures. 

The segregation and storage of protein reserves is obvious morpho- 
logically and the materials which are stored can be identified by adequate 
cytochemical methods. All the five Protozoa, again with the exception 
of Flabellula, have visible stores of proteins or derivative substances. 



172 CYTOPLASMIC INCLUSIONS 

A. pro feus forms leucine and glycine crystals in the gastrioles (they are 
therefore not strictly speaking cytoplasmic bodies), and these are then 
separated from the gastriole and the materials are transported from the 
crystal vacuoles to the growing refractive bodies and are there stored 
in the form of the protein stroma of these lipoid-protein bodies. In 
Opalina proteins are stored in the ectoplasmic segregation bodies. In 
both these cases, the final structures are of cytoplasmic origin and al- 
though they are so different they could perhaps be harmonized on a 
functional basis. In Ichthyophthkius, on the other hand, the protein 
spherules are stored and utilized in the cytoplasm, but originate in the 
macronucleus by splitting from the chromatin a portion which contains 
nucleic acid and iron, leaving a reserve protein in the form of large gran- 
ules which are then discharged as completed bodies into the cytoplasm. 
There is some evidence in the Sporozoa also of a nuclear origin of some 
of the protein reserves, although it is entirely possible that they are 
connected with mitochondria, since Joyet-Lavergne noted a morpho- 
logical relationship between the two in the Sporozoa he studied. How- 
ever, even if we disregard the somewhat questionable case of the Sporo- 
zoa, we find that an identical function — the storage in the cytoplasm of 
proteins — is accomplished in two cases by cytoplasmic structures, but in 
a third case by the macronucleus. 

Digestion, except in the astomatous species, is accomplished by the 
gastriole, a structure formed by the union of a vacuole which contains 
the food particles with granules or vacuoles of cytoplasmic origin. In 
Ichthyophthirius the granules involved are cytoplasmic in origin, but 
become enclosed within the vacuole; the cytoplasmic vacuoles of ¥la- 
hellula apparently furnish the fluid in the gastriole; while in A. proteus 
the granules merely aggregate around the gastriole. From morphological 
evidence, the granules in Ichthyophthirius and Flabellula are concerned 
with all types of digestion, but in A . proteus both morphological and mi- 
croenzymatic studies show that the mitochondria are concerned with the 
digestion of carbohydrates and with the transport of digested materials 
from the gastriole to such bodies as the refractive granules. There is 
thus some variation in the digestive function, but clearer evidence of 
differences is the fact that in both Ichthyophthirius and Flabellula the 
diffusion of materials outward from the vacuole is accomplished without 
the intervention of any visible granules. The granules of Amoeba, there- 



CYTOPLASMIC INCLUSIONS 173 

fore, have a transport function, but none of the granules of either 
Ichthyophthirius or Flabelliila give evidence of a relationship with the 
gastriole which would permit such a function. 

The storage of lipoids is accomplished in almost identical fashion in 
Opdina, Ichthyophthirius, and Gregarina by the formation of inter- 
mediate lipoid bodies which are converted into neutral fats. A. proteus 
also stores neutral fats, but in this case no visible intermediate bodies are 
formed. This is not necessarily in conflict with the facts observed in the 
other species, since it is very possible that the intermediate bodies of 
fatty acid might be present but never get as large as the lower limits of 
microscopic visibility. However, not all the visible lipoid reserves of 
Amoeba are in the form of neutral fat; some are in the form of masked 
lipoids in the refractive bodies, and none of the other Protozoa in this 
group have granules which are strictly comparable with these complex 
structures. 

Carbohydrate reserves are found in the form of paraglycogen gran- 
ules in Ichthyophthirius and the Sporozoa, and in both cases are secreted 
by mitochondria. In Opalina, glycoprotein is found in certain cases in the 
segregation apparatus, but according to Kedrowsky, there are no im- 
portant stores of carbohydrates in this species. For Amoeba likewise this 
statement holds, the only carbohydrate being the shell between the fluid 
and the lipoid-protein rim of the refractive bodies. No carbohydrate 
reserves are found in Flabellula. From these cases it would appear that 
the function of carbohydrate storage is largely accomplished by mito- 
chondria, but it must be remembered that this is not a general rule, since 
these reserves may be formed by independent bodies, as in the Ophryo- 
scolecidae, or by the parabasal body, as in certain flagellates. 

The vacuolar apparatus performs at least two functions — the excretion 
of water to maintain the proper water balance, and the excretion of 
other materials which are probably metabolic wastes. The first function 
may be performed without the intervention of granules, as in the encysted 
stages of Ichthyophthirius, but the excretion of other materials is accom- 
panied by the periodic aggregation of granules around the vacuole in 
Amoeba and Ichthyophthirius. In the former function the granules con- 
form to the definitions of mitochondria, and in the latter to the Golgi 
bodies. These bodies are certainly a group having to do, in Ichthyoph- 
thirius, only with the contractile vacuoles; but in Amoeba they are appar- 



174 CYTOPLASMIC INCLUSIONS 

ently also associated with the food vacuoles, the refractive bodies, and so 
forth, in a general transport function. The two groups are comparable, 
but the granules of Ichthyophthirius perform only a part of the functions 
assigned to them in Amoeba. It may be possible that the mitochondria 
or beta granules of Amoeba include more than one type, but in view of 
the detailed experiments which have been made on this form, this explan- 
ation is hardly more than a possibility. 

This survey of functions which have a granular basis fails to reveal 
any general uniformity, even in this restricted group of five Protozoa. 
The most clear-cut case of two different mechanisms having the same 
function is that of the segregation of reserve protein in Ichthyophthirius 
and Opalina. No matter how broad the definitions are made, the fact 
remains that the identical result is attained through the mediation of two 
different cellular mechanisms — in the one case the mechanism is the 
macronucleus, in the other it is strictly cytoplasmic, the segregation 
bodies. The general concept of transport introduced by Doyle helps in 
several cases to group apparently diverse functions within a single func- 
tional concept; but here again none of Kedrowsky's published obser- 
vations in the case of Opalina, nor my own observations in Ichthyoph- 
thirius, would support this. The stored fat, paraglycogen, and proteins 
merely decreased in size during encystment, and no intermediate bodies 
aid in the redistribution of this material. 

The granules which are produced in a particular species are typical of 
that species, but in other species the same function may be accomplished 
by granules different in composition and relationships from those of the 
first species, or the same function may be accomplished without the form- 
ation of visible granules. The cell is not restricted in the accomplishment 
of its functions by any system of universal and invariable cytoplasmic 
components. 

The Continuity of Cytoplasmic Granules 

The failure to find evidence of universal cytoplasmic components by 
the use of either composition or function as criteria, brings the discus- 
sion to a much more general concept — the distinction between granules 
as permanent organelles and as temporary reserve granules. This dis- 
tinction can be traced back to Altmann's bioblast theory, but it has been 
applied more recently in a modified and refined form to the mitochondria, 



CYTOPLASMIC INCLUSIONS 175 

Golgi bodies, and the vacuome. The rejection of the universahty of any 
one of these components still leaves the possibility that in each cell there 
are two sets of granules — the group which is permanent in organization, 
and a group of temporary granules, usually "passive" reserve bodies, 
which may be derived from the activities of the first. This is not a 
restatement of Altmann's theory, in the sense that it implies that the 
first group are living units as such, nor even that these granules are 
regarded in any strict sense as independent, since their maintenance 
obviously depends upon their interaction with the other parts of the 
cell. The question is whether any set of granules are present during the 
whole life cycle, and further whether new granules of the same group 
arise directly from the old granules and never arise de novo. 

The morphological studies of Faure-Fremiet (1910), Joyet-Lavergne 
(1926a), MacLennan (1934), Subramaniam and Ganapati (1938), and 
others have shown that mitochondria, Golgi bodies, glycogen granules, 
and so forth, in various Protozoa, undergo division in such a manner as 
to retain the original organization of the granules, and that these bodies 
are found in all stages of the life cycle. On the other hand. Horning 
(1929), Volkonsky (1929 on), MacLennan (1936), and Kedrowsky 
(1931 on) find that one or more of the supposedly fundamental com- 
ponents arise de novo either continuously or at some stage of the life 
cycle. A de novo origin has been proved by Mast and Doyle ( 1935b) not 
only for granules which are simple morphologically, but even for the 
complex tripartite refractive granules of A. proteus. In Ichthyophthirius 
apparently none of the granules are retained through the life cycle, thus 
clearly eliminating in this protozoan any distinction based on continuity. 
These morphological studies show that there may be a genetic continuity 
with respect to some granules in some of the Protozoa, but that it is not 
a general thing. 

The observation that in some Protozoa all of the granules arise de 
novo at some time or other, raises the question whether the observed divi- 
sions are significant or are merely incidental. Kedrowsky was able to in- 
duce typical division figures in the endosomes of Opalina by altering the 
culture medium. Horning (1929) showed that dividing mitochondria 
are found m the trophozoite of Monocystis but that these granules dis- 
appear completely during the spore stages and form de novo in the 
newly liberated sporozoite. The digestive granules of Ichthyophthirius 



176 CYTOPLASMIC INCLUSIONS 

divide inside the gastriole to form a larger number of small granules, 
and both the mitochondria and the intermediate lipoid bodies (endoplas- 
mic Golgi) fracture and split into rods as the granule within the sphere 
grows; yet in none of these cases is the division more than an incident 
in the cycle of the original granule, which arises in all cases de novo. The 
division of cytoplasmic granules is merely an indication that the granule 
is unstable under the particular conditions of size, surface tension, and 
so forth. 

The term "vacuolar reaction" was introduced by Volkonsky to de- 
scribe the relationship between the formation of new digestive granules 
(his vacuome) and the presence of food. The pattern of the reaction 
depended both upon the species of cell and upon the type of food present. 
This formation of granules as a response to a specific stimulus is by no 
means restricted to the single case of the digestive granules. The forma- 
tion of secretion granules in the Tintinnidae is a specific response to the 
factors which require a new lorica, and these granules are present at no 
other time. The formation of the complex refractive bodies in Amoeba 
is a specific response of that particular protozoon to excess food; when 
this condition no longer holds, the granules are resorbed. The excre- 
tory granules of Ichthyophthhius are the response of this protozoon to 
the presence of metabolic wastes, which result from active feeding and 
growth and which disappear in encystment, when the original condition 
no longer holds good. The segregation bodies of Opalina likewise exhibit 
changes which are specific responses to the particular food which is 
available. Horning (1929) points out that the disappearance and re- 
appearance of mitochondria in Monocystis is correlated with the decrease 
and increase of metabolism resulting from encystment and excystment. 
Volkonsky's vacuolar reaction is one case of general response, or "granu- 
lar reaction," of the cell to a host of stimuli. If the stimulus is always 
present, the particular granules which are characteristic for the stimulus 
and for the particular cell under consideration are always present, but if 
the stimulus is intermittent, the particular granules involved are present 
only for the corresponding period. 

Continuity is of no significance in the evaluation of the granules, but 
is rather a criterion of the continuity of the stimulus which induces the 
formation of the granule. This, together with the demonstration that 
the division of the granules is purely incidental, shows that it is not possi- 



CYTOPLASMIC INCLUSIONS 177 

ble to distinguish between permanent organelles and temporary com- 
ponents, nor between active and passive granules. 

The Classification of Cytoplasmic Granules 

The cytoplasmic granules are a visible reaction of the cell to various 
stimuli, with the result that they show as great a variety as do the func- 
tions of which they are the visible expression and as the cells which form 
these granules. Any final classification must be based, then, on function, 
composition, and origin, rather than on a few nonspecific stains which 
give the impression of universal components, or on a distinction between 
permanent organelles and temporary reserves. Since function, composi- 
tion, and relationship vary widely from one cell to another, the cyto- 
plasmic granules, even of the Protozoa alone, cannot be divided into 
three or four sharply defined types, but must be separated into more 
types, with a classification sufficiently flexible to allow for the combina- 
tion of several functions in the same granule. Such an ideal classification 
may be defined briefly as functional. 

A functional classification is impossible at the present time, since the 
usual cytological or cytochemical methods reveal only those functions 
which result in the accumulation of visible masses of material — the 
segregating functions. The general type of material which is segregated 
has been identified in many cases, but usually there is insufficient evidence 
to determine whether this is a simple segregation process or whether 
there is actual synthesis involved. A functional classification on such a 
narrow base would lack permanent value, but it is necessary to readjust 
the present classification, in order to separate granules which are obvi- 
ously unlike, even on the relatively scanty evidence now available. This 
separation has been outlined in the previous sections with the detailed 
evidence, but it is worthwhile to assemble these suggested changes here 
in one place. 

The mitochondria are those granules which respond to mitochondrial 
methods, such as those employed by Regaud, Benda, and so forth, and 
which usually segregate Janus green. This is admittedly a heterogeneous 
group, but there is insufficient information at present to separate any 
groups on a logical basis. 

The term Golgi body is used to designate granules or structures which 
impregnate specifically with the classic reduction methods, but excepts 



178 CYTOPLASMIC INCLUSIONS 

those bodies which are mitochondria, or which segregate neutral red 
vitally. The term as used here is thus merely a convenient way to indicate 
briefly certain techniques. Included in this group of granules are the 
fatty acid bodies, which are simply a stage in the formation of neutral 
fat granules and which have been called intermediate lipoid bodies. The 
granules or membranes which are associated with the contractile vacuoles 
are a separate group in composition and function, and are called excre- 
tory granules. There is a third group which display a characteristic secre- 
tory cycle but which are neither lipoidal in composition nor excretory 
in nature (Ellis, 1937). For these bodies and other unknown granules, 
the term Golgi body is appropriate, since it merely designates them ac- 
cording to the techniques used and implies nothing as to their composi- 
tion or functions. 

The term neutral red granule refers to any body which segregates 
neutral red or similar basic dyes in the living normal protozoon. Again, 
it is a term which indicates only the technique used and is a convenience 
when there is no evidence as to function. In this group are the segrega- 
tion granules, which accumulate and perhaps synthesize proteins and 
similar materials, as in Opalina. The refractive bodies of Amoeba may 
be included here because of the neutral red reaction and because of the 
protein stroma, or they could be listed in the lipoid reserves because of 
their high lipoid content. The digestive granules are a separate group 
and are associated with the gastriole. One of the problems in this con- 
nection is how these digestive granules differ in function and composi- 
tion from the mitochondria, which may also be associated with digestion. 

Many different types of granules, some of them with the power to 
segregate neutral red, are expelled from the cell in the formation of 
shells, cysts, cement, and so forth, and are named secretion granules. 
These granules should be given more attention, since they indicate a 
situation similar to the secretion granules of gland cells. 

The reserve bodies have been separated on the basis of the material 
stored — protein, lipoid, and carbohydrate — which also allows for the 
various combinations which do occur, and which will permit further 
subdivision when justified by an increase in the precision of cytochemical 
methods. This is convenient in summarizing the reserves, but for final 
classification it is unsatisfactory, since it ignores the differences in origin — 
whether they are independent bodies, as the segregation granules of 
Opalina and the paraglycogen granules of Poly plastron, or whether they 



CYTOPLASMIC INCLUSIONS 179 

are proHucts of the macronucleus, mitochondria, and so forth. This is a 
difficulty which cannot be overcome until the fundamental processes 
which are involved have been worked out. 

In addition to these granules are a heterogeneous group of unknoivn 
granules such as the alpha granules of Amoeba, and the accessory bodies 
formed by the neuromotor system of Haptophrya (Fig. 70); various 
pigment granules, which may in some cases be part of the lipoid reserves, 
or in some cases residues of food, as in Plasmodium and Ichthyophthirius, 
this latter type of course not being true cytoplasmic granules. Crystals 
also are often present, the ones in Amoeba being classed as a part of 
the protein reserves (although here there is a question, since they orig- 



Figure 70. Accessory 
bodies being formed from 
the neuromotor ring in 
Haptophrya m'tchiganens'is, 
Zenker's haematoxylin. 
(After Bush, 1934.) 



inate in the food vacuoles) on the evidence of Mast and Doyle. Other 
crystals, according to Reichenow, may be excretory granules. 

Comparison with Cells of the Metazoa 

Cytological investigations in the Protozoa have always been influ- 
enced by the transfer of concepts originally developed from a study of 
the cells of the Metazoa, particularly the vertebrates, with the result that 
the division of granules into mitochondria, Golgi bodies, vacuome, and 
passive reserve bodies are as common in the literature of the Protozoa 
as in that of the Metazoa. In spite of Dobell's denial of the cellular 
nature of the Protozoa, any consideration of the granules of the Protozoa 
necessitates a comparison with the cytoplasmic granules in other animal 
cells. 

The lack of a "typical" reticular net of Golgi in the Protozoa, the 
infrequency of filamentous mitochondria, and other striking morpho- 
logical differences between the protozoan and metazoan cells have been 
stressed so often that it is well to present several cases of equally striking 
similarities in both structure and function. Volkonsky (1934) found 




180 CYTOPLASMIC INCLUSIONS 

that the digestive granules and gastrioles of Protozoa, choanocytes, and 
leucocytes are entirely comparable, and included all of these cells in his 
vacuolar reaction. Faure-Fremiet (1909) and later Kedrowsky (1932, 
1933) compared the segregation granules of Protozoa and vertebrate 
tissues and found they were similar in appearance, staining reactions, and 
function. Chatton, Parat, and Lwow (1927), on the basis of specific 
microchemical reactions, compared the protein reserves in certain of 
the Foettingeriidae with the vitellin of hen's eggs. Kedrowsky (1931) 
and MacLennan (1936) have given figures of the formation of neutral 
fat bodies in the Protozoa which are almost identical with the descrip- 
tion and figures of Bowen (1929) in the relationship between Golgi 
bodies and lipoid secretion in cells of the mouse. Examples could be 
multiplied, but these are sufficient to emphasize the fact that there are 
similarities as well as differences between protozoan and metazoan cells, 
with respect to their cytoplasmic granules. 

The same difficulties with respect to the so-called specific staining 
reactions arise in both Protozoa and Metazoa. In a study of echinoderm 
eggs, Tennent, Gardiner, and Smith (1931) showed that not one ma- 
terial, but many reduce osmium in the Golgi techniques. The presence 
of more than one type of osmiophilic granules has been proved by 
Mast and Doyle (1935) and MacLennan (1936) in Protozoa. Although 
Kirkman and Severinghaus (1938) hold to the idea of a particular 
Golgi substance, they bear witness to the occurrence of additional osmio- 
philic materials: "One often finds small osmiophilic granules of uncer- 
tain significance in Kolatchev sections, but they are present in addition 
to the Golgi apparatus and appear to bear no relation to the latter struc- 
ture." In neither group is there any evidence that there is a particular 
Golgi substance, any more than a particular Golgi structure. 

There is also at times an embarrassing overlapping in the results ob- 
tained from the use of Janus green and neutral red. These dyes were 
found by Hopkins (1938) to stain the same vacuoles in the marine 
amoeba, Flabelhda, and Uhlenhuth (1938) reports similar results in 
the thyroid cells of amphibia. The mitochondria turn out to be not a 
simple group but a complex group, as indicated by the distinction 
between mitochondria and active mitochondria, or mitochondria proper, 
by Parat (1927) and by Joyet-Lavergne (1926). 

Evidence is accumulating in both Protozoa and Metazoa that no type 
of cytoplasmic granule (with the the exception of the centriole) can be 



CYTOPLASMIC INCLUSIONS 181 

considered as permanent, self-perpetuating structures. Wilson and Pol- 
lister (1937), in connection with an investigation on sperm formation 
in scorpions, review the division and distribution of mitochondra, Golgi 
bodies, and vacuome, and show that the supposed accurate division is 
actually an incidental fragmentation of large masses and that the distri- 
bution is random during the division of the cell. They state: "There is, 
however, little ground for the contention that either Golgi bodies or 
chondriosomes can be regarded as permanent individuals having the 
power of self perpetuation by growth and regular division." Exactly 
the same situation has been shown in this discussion with respect to the 
cytoplasmic granules of the Protozoa, from studies of both fixed material 
and of living normal cells. The permanence of mitochondria in metazoan 
cells has been summed up recently by Bensley (1937) : "The disappear- 
ance and reappearance of mitochondria in living cells under observation, 
as described by Chambers, however repugnant the idea may be to those 
who would elevate mitochondria to the dignity of living, self-reproduc- 
ing units, must be definitely entertained as probable." With respect to 
the de novo origin of Golgi apparatus, Kirkman and Severinghaus 
(1938) assert that "there is little to favor such a view," although they 
quote at least a dozen authors who have advanced evidence of a de novo 
origin in one form or another, admitting in several cases that the evidence 
presented has not been refuted. For a detailed discussion, the reader is 
referred to the review of the subject by these authors and to the original 
publications, but it is clear that not all cytologists agree with these authors 
on the permanence of the Golgi bodies. 

The concept that the cytoplasmic granules arise or are resorbed as a 
result of specific conditions of metabolism in the cell — in other words 
that there is a granular reaction — is a logical result of the evidence that 
the granules are neither permanent nor self-perpetuating, and it is 
therefore no surprise to find that this interpretation has been made with 
respect to the cytoplasmic constituents of the Metazoa as well as of the 
Protozoa. One of the clearest statements of this concept has been made 
by Tennent, Gardiner, and Smith (1931) : "The results of this research 
have been the conviction that neither Golgi bodies nor Chondriosomes 
are structural elements in the cellular architecture, but that both are 
the chemical products of physiological processes." Nahm (1933) like- 
wise states that "they are the visible products of chemical reactions that 
occur in the cell." 



182 CYTOPLASMIC INCLUSIONS 

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CYTOPLASMIC INCLUSIONS 187 

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'Paramecium caudatum. Arch. Protistenk., 51: 184-88. 
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■ 1929. Doflein, Lehrbuch der Protozoenkunde. 5th ed., Jena. 

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C. R. Soc. Biol. Paris, 107: 611-14. 

1931b. Coloration vitale et potentiel d'oxydo-reduction chez les gre- 
garines. C. R. Soc. Biol. Paris, 107: 1508-11. 



CYTOPLASMIC INCLUSIONS 189 

Rosenberg, L. E. 1937. The neuromotor system of Nyctothenis hylae. Univ. 

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190 CYTOPLASMIC INCLUSIONS 

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CHAPTER IV 

FIBRILLAR SYSTEMS IN CILIATES 
C. V. Taylor 

Introduction 

The essential nature of Leeuwenhoek's "little animals" remained 
obscure for more than 150 years, evidently because the methods of ob- 
servation which characterized that ingenious microscopist of Delft were 
replaced largely by fruitless speculation. Otherwise, man's epochal dis- 
covery of the cellular nature of living things might have been realized 
sooner. 

Meantime, it is true, a prodigious diversity of macroscopic forms had 
been examined and classified. But the disclosure of cellularity, which 
eventually unified all of this diversity in organic form, had to depend 
upon the detailed analysis of organic structure. 

During the hundred years that have now intervened, that common 
denominator of organic form and function has come to be regarded, for 
multicellular plants and animals, as a sort of master key to the solution 
of their fundamental problems. And for the major advances in biology 
during that memorable century, we are surely indebted primarily to this 
cellular concept of the organization of living things. 

For the microorganisms, however, the concept of cellularity, although 
generally conceded, has encountered not infrequently some confusing 
difficulties. With von Siebold's pronouncement in 1845 of the unicellu- 
larity of the Protozoa, the way at first seemed clear toward simplifying 
and unifying all forms of life, in terms of the cell as the universal 
unit. Eventually, however, it was evident that, for the Protozoa, this 
concept did not simplify matters so satisfactorily. The chief difficulty 
here arose in trying to equate the protozoon cell with a tissue cell of 
the Metazoa. And even in recent times this comparison has again been 
challenged by Dobell (1911) and others, who would maintain that 
Protozoa are not cells at all and so should be regarded as non-celluar 
organisms. 



192 CILIATE FIBRILLAR SYSTEMS 

But similar difficulties in comparing microscopic with macroscopic 
forms of life had confronted investigators several years before von Sie- 
bold's pronouncement and, in fact, before the concept of cellularity had 
been definitely formulated. As is well known, this all culminated in 
the Ehrenberg-Dujardin controversy, beginning in 1835- Obviously for 
these investigations, the issue was not one of cellularity, but it had to 
do with complexity versus simplicity in the organization of the Infusoria. 
It seems probable that Ehrenberg defended his thesis of "complete or- 
ganisms" partly in refutation of the theory of spontaneous generation, 
then vigorously championed for microorganisms. At any rate, he sought 
to identify in the Infusoria all the organs common to other animals. 
Much of his adduced evidence, it will be recalled, was successfully re- 
futed by Dujardin, who described among other things, his newly dis- 
covered "sarcode" in support of his contentions for uniqueness and sim- 
plicity in the organization of the Infusoria. 

The essentials of these contrasting views of Ehrenberg and Dujardin 
on the nature of infusorian organization have recurred, in varied guise, 
many times in the literature since their day. These opposing viewpoints 
have, of course, become translated into terms of the concept of cellu- 
larity, so that now the nature of unicellular organization, or "proto- 
plasmic differentiation," is commonly contrasted with "cellular differ- 
entiation" of multicellular organisms. 

Accordingly, in the following review of literature on fibrillar systems 
in ciliates, it will become evident that some discrepancies in both the 
analysis of structure and the interpretation of functions may owe their 
origin largely to contrasting points of view on the essential nature of 
"protoplasmic differentiation" in the Protozoa and "cellular differentia- 
tion" in the Metazoa. 

Before beginning that review, however, the fact should be emphasized 
that, as Maupas (1883) has pointed out, the Ehrenberg-Dujardin con-, 
troversy marks a turning point in protistological investigations. Not only 
did it enlist a wider interest in these microorganisms, but it made clear 
the necessity of a critical structural analysis of their greatly diversified 
types of organization and of a comparative study of such types before 
any satisfactory interpretations were possible. 

The literature resulting from those analyses is so voluminous that 
when one undertakes to review the accounts of a given system of organ- 



CILIATE FIBRILLAR SYSTEMS 193 

elles, such as the fibrillar system, and to condense that review within 
reasonable bounds, the difficulties soon become evident. For this reason 
it has seemed advisable in the review that follows, in the interests of 
students and laity as well as of specialists, to present a fairly detailed 
account of the structural analysis, together with interpretations of the 
fibrillar differentiations of a well-known representative of each of four 
major groups of ciliates. This is followed by a brief review of other 
published work, mostly since 1920, on fibrillar systems in other ciliates, 
with some suitable illustrations; and finally, a few paragraphs of general 
discussion are added under the caption "Conclusions." 

The discussion of the structural analysis of the fibrillar systems of the 
four representative ciliates, Paramecium, Stentor, Euplotes and Vorti- 
cella, is off^ered first and separate from the interpretations for these four 
ciliates, whose order is then, for convenience, reversed. This separate 
treatment was decided upon primarily for the sake of accuracy and clarity. 
Often in the literature the author's interpretations are so intermixed with 
his factual descriptions that it is sometimes very difficult to make certain 
just what he observed and undertook to describe. 

Examples of Fibrillar Systems 

a. structural analysis 

1. Paramecium. — This familiar representative of the holotrichs has 
doubtless been more generally used in both teaching and research labora- 
tories than has any other of the numerous kinds of ciliates. Probably 
its apparent simplicity, more than its smaller size, tended to discourage 
a search for a fibrillar system, such as had been found in Stentor and 
other forms. 

In 1905, however, Schuberg described for both Paramecium and 
Prontonia fibrillar diff^erentiations which, running close under the pel- 
licle, united the basal granules in the longitudinal rows of cilia. By 
means of a bichromate-osmic fixative and Loeffler's stain, not only was 
this relationship of fibril and basal granules clearly defined, but also, 
because of their staining properties, they could be well differentiated 
from the hexagonal, or rhomboidal, pattern of the pellicle, as was well 
illustrated in Schuberg's several figures. 

In 1925, J. von Gelei described in Paramecium nephridiatum a periph- 
eral network of fibrils which was not connected with the familar po- 



194 CILIATE FIBRILLAR SYSTEMS 

lygonal pattern observable in the living organism. For fixation, he used 
Apathy's sublimate-osmium and stained with toluidin blue. 

In the following year Klein (1926a) reported the results of his 
studies on a peripheral fibrillar complex in certain ciliates by means 
of a new silver-nitrate technique which involved no previous fixation. 
The method, thus employed, is now well known as the "dry method," 
in contrast to von Gelei's (1932a) "wet method," and the resulting 
silver-impregnated fibrillar complex is quite commonly referred to as the 
"silverline system." 

The several subsequent publications of these two authors on the fibril- 
lar system of Paramecium, using especially the silver-nitrate technique 
but also other methods, admit of useful comparisons for this brief re- 
view, so that their results will now be considered together. 

In most of these various articles, the author's account of the structures 
that were clearly observed is at times so involved with his avowed inter- 
pretations of their functions that it has been found difficult to sift out 
the essential data for which this review is intended. 

Their structural analyses of the fibrillar system of Paramecium have, 
nevertheless, several important points in common which may now be 
fairly, and as simply as possible, presented. On the basis of these com- 
mon points, certain discrepancies will then be indicated. 

To this end, it will be convenient to recall the findings of Schuberg 
(1905). He observed (1) a differentially stainable pellicular pattern, 
which was hexagonal over the body and rhomboidal on each side of 
the mouth, and below this {2') a longitudinal fibril connecting (3) the 
basal granules in each row of cilia — each such granule appearing below 
the center of each pellicular polygon (Fig. 71 ) . 

In outline, the descriptions of both von Gelei and Klein present this 
same general picture, which may now serve to simplify a brief com- 
parison of their essential findings. In their later papers, both of these 
authors agree that the fibrillar system of Paramecium is entirely subpel- 
licular. Bearing this in mind, we may note that: 

1. Schuberg's pellicular pattern corresponds in general outline to 
von Gelei's "Stiitzgitter System" and to Klein's "Indirekt verbindung 
System" ("Meridiaan II. Ordnung"). 

2. Both the Stiitzgitter System and the Indirekt verbindung System, 
lying under the pellicle, comprise each: ( 1 ) a longitudinal fibril, between 



CILIATE FIBRILLAR SYSTEMS 195 

the rows of cilia; (2) cross fibrils, connecting adjacent longitudinal 
fibrils; (3) opening for a trichocyst midway on the cross fibril; and (4) 
a suture line at the anterior and the posterior poles. The resulting lattice, 
therefore, has its counterpart in Schuberg's pattern. 

3. Beneath von Gelei's Stiitzgitter System lies his "Neuronem Sys- 
tem." 

4. Beneath Klein's Indirekt verbindung System lies his "Direkt ver- 
bindung System." 

5. Both Neuronem System and Direkt verbindung System comprise 




\\ \ 






■w 



Figure 71. Pellicular Pattern and Longitudinal Fibrils Connecting Basal Granules in 
Paramecium. (Modified from Schuberg, 1905.) 
b. gr.^ — basal granule 1. fib. — longitudinal fibril pell. p. — pellicular pattern 

each: (1) an interciliary fibril, connecting the "Basalapparaten" in each 
longitudinal row of cilia; (2) cross fibrils, connecting the interciliary 
fibrils; and (3) Relations korner, which include the Basalapparat and the 
Trichocystenkorn . ' ' 
The interciliary fibril and the Basalapparaten, therefore, find their 
counterpart essentially in Schuberg's longitudinal fibril and its connected 
basal granules. 

For both von Gelei and Klein, accordingly, it is evident that the 
fibrillar system of Paramecium includes two subsystems, or fibrillar com- 
plexes. For present convenience and especially for later discussion, I 
shall refer to these as von Gelei's outer fibrillar complex and inner fibril- 
lar complex and Klein's outer fibrillar complex and inner fibrillar com- 
plex respectively. 



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CILIATE FIBRILLAR SYSTEMS 



197 



The several components of these fibrillar complexes, which were listed 
above, constitute the main structural features shared by Klein and von 
Gelei in their various accounts of the fibrillar system of Paramecium. 
But certain discrepancies appear in their descriptions of these and espe- 
cially of some other components. 

Space does not permit a discussion of all of these discrepancies, but 
most of them will be found listed in Table 1, which comparatively sum- 
marizes all the structural components ascribed to Parameciu7n's fibrillar 
system by von Gelei and by Klein in their various publications. Some of 




tri. 



Figure 72. Diagram of G'ttter (lattice) with attached trichocysts and of the neuro- 
nemes connecting bases of the cilia. (Von Gelei, 1925.) 
Git. — Gitter neu. — neuroneme tri. — trichocyst 



the discrepancies may very possibly be attributable to species differences, 
since von Gelei studied P. nephyidiatum and P. caudatum, while Klein's 
descriptions are of P. aurelia. 

A few discrepancies should, however, here be pointed out. The first 
and most important of these has to do with any structural integration 
between the two fibrillar complexes. Klein especially emphasizes the fact 
that his outer fibrillar complex and inner fibrillar complex are a con- 
tinuum through interfibrillar connectives. Von Gelei, on the contrary, 
denies that any connection exists and so states that his outer and inner 
fibrillar complexes are only contiguous (Fig. 72) . 

A further discrepancy concerns the basal apparatus of the cilium. 
According to von Gelei (1932a), this apparatus consists of (1) a basal 



198 CILIATE FIBRILLAR SYSTEMS 

ring surrounding (2) the basal granule of the cilium, and (3) the 
"Nebenkorn" occurring at the right of the junction of the basal ring 
and the interciliary fibril (p. 158). According to Klein (1931), the 
elements of the basal apparatus include (1) a ring (Zirkularfibrille) 
within which are usually three granules (Drierkorner). The central 
granule is the basal granule, and the other two are Nebenkorner. The 
discrepancy here is not only in the relative number of granules in the 
basal apparatus but in their relationship, since von Gelei regards his 
"Nebenkorn" as identical with Klein's basal granule, and accounts for 
Klein's third granule as being only a thickening of the basal ring. 

Finally, mention may be made in this connection of an additional 
system of fibrils described by Gabor von Gelei (1937) in three species 
of Parameciujn, — P. caudatum, P. multhnkronucleata, and P. trichinum. 
This third fibrillar complex, in addition to the two noted above, was 
found at the level of and below the basal granules. Its fibrils spread 
throughout the entire body surface, including the vestibule, where it 
sends a thickened fibril into the cytopharynx between the membranelles. 

The general pattern of this complex, made up of longitudinal and 
cross fibrils, resembled that of the outer fibrillar complex, the meshes 
of the former being smaller and more numerous, however, than those 
of the latter. Also, the course of the fibrils of this third complex were 
more irregular. Its longitudinal fibrils become fewer in the middle region 
of the body and at times are directed diagonally, even spirally. A splitting 
of fibrils was occasionally observed, as well as variations in their thick- 
ness. They may also anastomose and form a "Schaumgiter." 

The author could discover no connection between this fibrillar complex 
and either of the other two. 

Apparently the most recent detailed account of the fibrillar system 
of P. caudatum, including that of its cytostome, was made by Lund 
(1933), working in Kofoid's laboratory. After comparing the descrip- 
tions of earlier workers (Engelmann, 1880; Maupas, 1883; Schuberg, 
1905; Rees, 1922; von Gelei, 1925-32; Klein, 1926-31; Jacobsen, 1931) 
with his own findings, Lund concluded that previous investigators had 
confused "parts of at least two and possibly three quite difi^erent aggre- 
gations of structures, namely, the pellicle, the trichocysts and the periph- 
eral portion of the neuromotor system. In addition" they had failed 



CILIATE FIBRILLAR SYSTEMS 199 

"completely to demonstrate the great pharyngeal complex," which is 
an integral part of this system. 

Lund was able to differentiate between these "different aggregations 
of structures" by means of the silverline technique (Klein's and von 
Gelei and Horvath's) on the one hand and, on the other, by the use of 
iron-haematoxylin and Mallory's stain. 

The former method demonstrated von Gelei's "Stiitzgitter System" 
and Klein's "Indirekt verbindung System," i.e., their "outer fibrillar 
complex" noted above. It also revealed essentially their "inner fibrillar 
complex," also as noted above. 

But, according to Lund, these are separate and distinct "aggregates." 
The "outer fibrillar complex" is not subpellicular, as both von Gelei 
and Klein maintain, but represents rather the sculptural polygonal pat- 
tern of the pellicle itself. A similar interpretation was made by Brown 
(1930). 

The "inner fibrillar complex" of von Gelei and of Klein comprises the 
basal granules, their connecting longitudinal body fibrils, transverse 
fibrils connecting the longitudinal fibrils, and others which include the 
"radial fibrils." These last mentioned "originate as longitudinal fibrils 
in the cytopharynx and oesophagus, spread radially out from the oral 
opening over the body surface and terminate a short way from the cyto- 
stome." 

This inner fibrillar complex may be clearly demonstrated by the silver- 
line techniques, especially by the wet method. There is, however, a 
portion of the fibrillar complex within the cytopharynx and cytoesopha- 
gus which is not wholly demonstrable by these techniques. This was well 
differentiated by the iron-haematoxylin and Mallory's methods, and 
described as "seven major parts, namely: (1) the pharyngo-esophageal 
network, (2) the neuromotorium, (3) the penniculus, (4) the oesopha- 
geal process, (5) the paraesophageal fibrils, (6) the posterior neuromotor 
chain, and (7) postesophageal fibrils." 

For the descriptive details of this very elaborate complex of fibrils 
and associated parts, obviously the original account must be read. It is 
evident, however, from this brief review of the results of these several 
workers on the fibrillar system of Paramecium that a number of discrep- 
ancies need to be cleared up and perhaps further structural analysis of 



200 CILIATE FIBRILLAR SYSTEMS 

this system made before we can hope to have a complete understanding 
of the parts that are, or are not, structurally integrated. 

In recent publications Chatton and Lwoff (1935, 1936) have de- 
scribed a fibrillar complex in several ciliates, which has long been known 
(Chatton and Lwoif, 1936) but has not been clearly distinguished from 
Klein's silverline system. The fibrils are visible in vivo and may be clearly 
difi^erentiated in preparations fixed in Bouin's or Champy's solution and 
stained in iron-haematoxylin. 

Each fibril [cinetodesjne) has connected, always along its left side, 
the basal granules {cinetosomes) of a longitudinal row of cilia. The 
fibrils, together with their adjoined basal granules (the so-called infra- 
ciliature), are each essentially an independent entity. They are never 
united by anastomosis or otherwise at either body pole, and so include 
no transverse or other fibrillar connectives throughout their course. 

The fibrils of this injraciliature are entirely superficial and adhere 
to the pellicle as rectilinear (never sinuous) threads. Other granules, as 
well as the ciliary basal granules, appear likewise attached, and these 
represent successive stages of the multiplication of the basal granules. 
Fibrils and granules stain alike, but in some species the fibrils cannot 
be impregnated with silver by the usual techniques. After fixation (osmic 
acid. Da Fano, Champy) and covering with gelatin or gelose, the fibrils 
may show, upon silver-nitrate treatment, the basal granules connected 
to a sinuous thread which, with its various connectives, represents Klein's 
silverline "plexus." This plexus, according to Chatton and Lwoff 
(1935), is acid labile and cannot be stained. 

The selective staining properties and relations of the infraciliature 
show that it is quite distinct from the silverline fibrils, and is comparable 
with the flagellar ridges of the Hypermastigidae, marking the place of 
formation and of the insertion of the cilia. 

It would appear that Chatton and Lwoff 's infyaciliature may be identi- 
fied with the longitudinal fibrils and basal granules of the inner fibrillar 
complex reviewed above. The left lateral attachments of the cinetosomes 
to the cinetodesmes is evidently a new finding. 

2. Stentor. — In his search for organs that would account for the 
well-known contractile behavior of Stentor, Ehrenberg (1838) saw in 
its conspicuous longitudinal bands the seat of that contractility. This 
interpretation of its contractile mechanism was accepted by several later 



CILIATE FIBRILLAR SYSTEMS 201 

investigators including Kolliker (1864) and, according to Neresheimer 
(1903), Haeckel's (1873) "Myophanen" should be so construed. 

It was between these longitudinal stripes, within the clearer non- 
pigmented meridians (Biitschli's "Zwischenstreifen"), that Lieberkiihn, 
in 1857, found a distinct contractile fibril coursing from the basal disc 
forward to the adoral zone. Greeff (1870) confirmed these findings 
and Engelmann (1875) made detailed studies on the refractive and 
contractile properties of the fibers which have come to be commonly 
referred to as myonemes. 

Four authors may be cited, among many others, for the descriptive 
details of the fibrillar system of Stentor: Schuberg (1890), Johnson 
(1893), Neresheimer (1903), and Dierks (1926). These have been 
the main sources for the following brief review of this system. 

Schuberg (1890) made several important observations on the arrange- 
ment of the myonemes of Stentor coeruleus and an analysis of the basal 
apparatus of the membranelles. He found that the course of the body 
myonemes, from the basal disc to the peristome border, was not con- 
stant. Instead, some showed bifurcations, with occasional re-branching. 
This branching of myonemes followed consistently a corresponding 
branching of the longitudinal rows of cilia and their adjacent, non- 
pigmented bands. Similar relations of bands, ciliary rows, and myonemes 
obtained also for the peristome field. 

Schuberg further observed that the double row of cilia, comprising 
a membranelle, was seated in an ectoplasmic basal platelet (""Basal- 
saum"), itself bipartite, below which appeared a triangular lamella 
("Basallamelle"). The inwardly directed apex of this triangle was 
continued as a fibril ( "Endf adchen" ) which was, in turn, united to all 
other such end fibrils by a basal fibril. The latter then ran rather deep 
below and parallel to the entire series of membranelles. 

Schuberg's account of Stentor's myonemes and his analysis of its 
membranelles have been generally confirmed, with the exception of the 
basal fibril. The latter was identified by Johnson (1893), Maier (1903), 
and Schroder (1906). But Neresheimer (1903) and Dierks (1926) 
are certain that, as such, it does not exist. It is worth noting that Schu- 
berg's "Basalfibrille" has been widely cited in the literature. 

Johnson's (1893) work is not concerned primarily with a structural 
analysis of Stentor's fibrillar system, but his observations were thorough 



202 CILIATE FIBRILLAR SYSTEMS 

and critical and, for the most part, they have remained vahd. Reference 
now may be made to his search for the so-called myoneme canal, de- 
scribed by Butschh and Schewiakoff (1889, p. 1297). 

Beneath the "Zwischenstreifen" they found a fairly spacious fluid- 
filled canal which surrounded the myoneme throughout its course. 
Johnson looked in vain for this canal, finally deciding that he was 
"unable to find the least evidence of such a structure, either in optical 
or actual sections." The majority of authors — including Delage and 
Herouard (1896), Maier (1903), Neresheimer (1903), and especially 
Dierks (1926) — agree with Johnson that the canal does not exist except 
as an artefact. Schroder (1907), on the contrary, affirms its form to be 
oval or circular in cross-section, its shape and position varying with the 
degree of body constriction. Roskin (1918) and von Gelei (1929) 
claim also to have definitely identified it. The latter regards it as an 
""organic part" of the myoneme, '"solid and elastic." 

Neresheimer's chief contribution to the microanatomy of Stentor was 
his discovery of another complex of fibrils to which he gave the name 
"Neurophanen." These were associated contiguously with the myonemes, 
but coursed usually peripheral to them. In suitable preparations which 
had been diflferentiated with Mallory's triple stain, the myonemes were 
distinctly red, whereas the neurophanes were colored a dark violet. The 
Zwischenstreifen remained unstained. Schroder (1906) maintained that 
these fibrils were rather only a structural feature of the "Zwischen- 
streifen," which, according to his results, also with Mallory's stain, did 
show an intense purple color. More recently, however, von Gelei (1925) 
and Dierks (1926) have identified similar fibrils, as will be noted 
further on. 

Neresheimer traced these neurophanes as coursing, each fibril directly 
over a myoneme, from the aboral plate to about halfway up the body. 
Here some ended in a knob and all others disappeared before reaching 
the peristome border. While the myonemes became shorter and thicker 
in fully contracted Stentors, the neurophanes appeared sinuous but 
otherwise remained unchanged. It is not clear, however, how Neres- 
heimer could make sure of the changed or unchanged appearance of 
these fibrils, since he stated that he was not able to fix Stentor in an 
uncontracted state. Evidently the myonemes may be visible in the living 
organism (Biitschli, 1889; Johnson, 1893), which may have been 



CILIATE FIBRILLAR SYSTEMS 



203 



Neresheimer's means of observing a "three-fold increase" in the thick- 
ness of the contracted myonemes. 

Perhaps the most complete structural analysis of the fibrillar differ- 
entiations of S. coeruleus is that by Dierks (1926). His work, which 
was carried out in Korschelt's laboratory, considerably revised and 
extended earlier accounts of the fibrillar system of this heterotrich. 

He noted a gradual thickening of the myonemes from the peristome 
border down to the aboral pole, where the fibers do not end abruptly, 



Figure 73. Connecting 
branch from neuroid to 
myoneme in Stentor. 
(Dierks, 1926.) 
myo. str. — striation of myo- 
neme 
neu. — neuroid 
neu. br. — neuroid branch to 
myoneme 



neu. — 




..neu. dr. 
_ _ myo. sir. 



as Johnson (1893) thought, but bend sharply inward and revert an- 
teriorly to form a pencil-like bundle (see also Schr5der, 1906a). This 
bundle soon becomes fimbriated, its component fibrils branch, and their 
tapering ends disappear in the cytoplasm "near the center of the con- 
tracted animal." 

Dierks confirmed Neresheimer's (1903) findings of a second fibril 
coursing parallel and usually peripheral to the myoneme, both of which 
also stained differentially by Mallory's method. But the relationship of 
these two sorts of fibrils was found by Dierks to be evidently more 
intimate than Neresheimer had observed. In various sectioned and 
stained preparations, the smaller fibril, or "neuroid," gave off one or 



204 CILIATE FIBRILLAR SYSTEMS 

more branches (Fig. 73) to its adjacent myoneme, with which it 
apparently united (cf. von Gelei, 1929b). 

He observed also the knob-hke endings of these neuroids, as described 
by Neresheimer for his neurophanes, but Dierks apparently could ac- 
count for such knobs as being merely the cross sections of fibrils that 
happened to be bent near the plane of section. 

The cross striations of the myonemes, described by Biitschli and 
Schewiakoff (1889), were observed by Dierks in the living organism 
as well as in his preparations. Johnson (1893) had regarded these as 
artifacts due possibly to wrinkling of the myonemes, but the regularity 
of their recurrence and spatial relations seemed to preclude this. The 
myonemes were usually elliptical in cross section, with the longer axis 
of the ellipse directed toward the center of the body. This cross section 
revealed definitely an outer cortex (Plasmahiille) and a medulla (Plas- 
mamark) (cf. Roskin, 1918). 

Dierks' analysis of Stentor's membranelle apparatus differs in several 
points from most earlier descriptions. The membranelle platelet, sup- 
porting each membranelle, represented essentially the aggregate of basal 
granules of the component cilia. ContinuinV from these granules into 
the cytoplasm was a basal lamella, the outline of which was clearly 
rectangular and not triangular as Schuberg (1890) had claimed for 
his "Basallamelle." Dierks' rectangular lamella could appear as a tri- 
angle, whose apex might be directed either toward or away from the 
basal granules, depending upon their position when viewed. For these 
lamellae were as ribbons, each about three times as long as broad, and 
each alike was slightly twisted on its long axis. This, according to Dierks, 
accounted for the erroneous interpretation of Schuberg ( 1890) , Schroder 
(1906), and others. Not only might the lamellae appear as triangles, 
but also their inwardly directed "apexes" might then seem to be con- 
tinued as a fibril ("Endfadchen"). To account for the basal fibril, which 
Schuberg thought united all of the end fibrils ("Endfadchen"), Dierks 
observed that his basal lamellae overlapped in such a way that their 
ends could give the impression of a continuous fiber, comparable in 
appearance, direction, and extent to Schuberg's described "Basalfibrille." 

3. Euplotes. — As a major group of ciliates, the hypotrichs probably 
mark the acme of highly differentiated motor organelles (undulating 
membranes, membranelles, and cirri) the related fibrillar system of 



CILIATE FIBRILLAR SYSTEMS 205 

which may appear correspondingly specialized. Another structural dif- 
ferentiation, the pellicle, assumes in this connection a significant im- 
portance in maintaining the flattened bodily form so characteristic of 
the hypotrichs. The often remarkable rigidity of this pellicle has long 
been recognized. 

Euplotes, the representative whose fibrillar system will now be 
reviewed, suitably illustrates these well-known characteristics of the 
hypotrichous ciliates. 

Maupas (1883) was apparently the first to identify fibrillar differ- 
entiations in this genus. He described in Euplotes patella var. a fibril 
extending anteriorly from the basal plate of each of the five anal cirri. 
These five fibrils united into a single fiber, which continued anteriorly 
and disappeared near the bases of the adoral membranelles. 

Maupas's findings were essentially confirmed by Prowazek (1903) 
in his brief account of protoplasmic reorganization in E. harpa. He 
further observed the "solide und fest" nature of fixed anal cirri fibrils, 
as indicated upon sectioning, when they might be pulled and bent 
thread-like by the microtome knife. Similar fibrils were seen to extend 
radially from the bases of the other cirri. Prowazek also described and 
figured still finer fibrillar lines ("Fibrillenziige") going in parallel to 
the adoral membranelles. These finer fibrils have apparently not been 
identified as such by later workers. 

Some years later Griffin (1910) gave a fairly detailed description 
of the fibrillar system which he discovered in E. worcesteri. From the 
base of each of its five anal cirri, he observed a fiber extending ante- 
riorly. All five fibers converged toward the adoral membranelles, near 
which they disappeared "close to each other." Unlike similar fibers 
described by Maupas (1883) and by Prowazek (1903) for other species 
of Euplotes, these of E. worcesteri apparently did not unite to form 
a common strand and were not traceable to the membranelles. Several 
finer fibers were found associated with the bases of some of the other 
cirri. Their number and direction varied, however, and they had no 
connection with those of the anal cirri. Griffin suggested that all of 
these fibers might be comparable to myonemes, the number of which 
had become reduced with a reduction in rows of cilia, as postulated for 
the hypotrichs generally; but he noted also that some of the fibers may 
be directed even transverse to the hypothetical original ciliary rows. 



206 CILIATE FIBRILLAR SYSTEMS 

Yocom (I9I8), working in Kofoid's laboratory, found and described 
in E. patella a fibrillar system much more extensive than that delineated 
in other Enplotes by Maupas, Prowazek, and Griffin, as noted above. 
In addition to the anal cirri fibers, such as they had found, Yocom 
discovered in E. patella "a fiber connecting the inner ends of the 
cytostomal membranelles" ("anterior cytostomal fiber"), and a "ino- 
tor'ium" (after Sharp, 1914) which united the membranelle fiber with 
those from the anal cirri. A structural integration was traced, therefore, 
between the cytostomal membranelles and the anal cirri. Similar fibers, 
radiating from the bases of the other cirri, were also described, but no 
connection was found between them and the others above mentioned. 
In the "anterior lip" of this species, Yocom depicted a fibrillar lattice- 
work which was united by "short rodlike projections" to the mem- 
branelle fiber. 

The intimate contiguity between this fibrillar system of E. patella 
and its motor organelles was clearly detailed by Yocom. Certain minor 
modifications and additions to his account were made by Taylor (1920), 
from studies especially of dissected and slowly disintegrating organisms. 
Following Sharp's (1914) terminology for a comparable fibrillar system 
which he had found and elaborately described in Diplodinium ecauda- 
tum, Yocom designated this system in E. patella a "neuromotor ap- 
paratus." 

The neuromotor apparatus discovered by Yocom is to be distinguished 
from an additional fibrillar system in this same species, which was 
carefully worked out by Turner (1933) by means of his modification 
of Klein's (1926) and von Gelei and Horvath's (1931) methods. His 
technical procedure is here worthy of note. After fixing the organisms 
in osmic acid vapor for about three seconds, and before the material 
was quite dry, Turner added two or three drops of 2-percent silver 
nitrate. Within four to eight minutes the nitrate was poured off and the 
slide placed in distilled water, barely covering the preparation. Over a 
white background, the slide was then exposed to the sun until the 
reduction of the nitrate had progressed as desired, according to occa- 
sional microscopic examinations. The preparation was then thoroughly 
washed in distilled water, dehydrated, and mounted. "The method gives 
strikingly clear-cut results." For this study, various other techniques 
were also employed, on both whole mounts and sections. 



CILIATE FIBRILLAR SYSTEMS 207 

By these several methods Turner was able to disclose an "external 
fibrillar network," which included: 

1. The "dorsal network," comprising (1) seven to nine longitudinal rows 
of granular rosettes, from each of which protruded a central bristle; (2) seven 
to nine longitudinal fibrils, uniting the bristles of the rosettes and designated 
primary fibrils; (3) longitudinal secondary fibrils, running midway between 
the primary fibrils; (4) commissural fibrils connecting, midway between the 
rosettes, the primary fibril and on either side its secondary fibril, "pulling the 
latter slightly out of line." The square meshes of this dorsal network, which 
appeared remarkably constant, averaged about four microns across. 

2. The "collar," anterior to this dorsal network, comprising, (1) the 
inclined row of parallel basal plates of the adoral membranelles, the lower 
ends of which "rest" on the oral lip; (2) a posterior membranelle fibril, 
connecting the "upper" ends of the membranelle basal plates; (3) an anterior 

post. m. fib. j(WFf'''^''^^^T^^^^(^^UWM^ 

mem, pi. -^^^gStJCTpiCSyCL' 

ant. m. fib. -^^^4^ H^Wrr / 




mec/. m. fib. ^^^^y^v-Jlrr^MiM^A^^^^ - - 3^1^- br. 

_-. ros. 

_ _ . com. ffb. 

JAJ^M^f^y^- sec. fib. 

-i-i—i—^--^'''^\ pri. fib. 

Figure 74. Euplotes patella: dorso-lateral view of external fibrillar system. (Turner, 
1933.) 

ant. m. fib. — anterior membranelle fibril com. fib. — commissural fibril 

med. m. f. — median membranelle fibril mem. pi. — membranelle plate 

post. m. fib.- — posterior membranelle fibril pri. fib. — primary fib. 

ros. — rosette sec. fib. — secondary fibril sen. br. — sensory bristle 

membranelle fibril, connecting short commissures at the "lower" ends of 
these basal plates; (4) a median membranelle fibril, attached on the basal 
plates between the anterior and posterior membranelle fibrils. 

These anterior, median, and posterior membranelle fibrils continue this 
same relationship with the membranelle basal plates throughout the course of 
the membranelles along the ventral "lapel" down to their ending in the 
cytopharynx (Fig. 74). 

Of primary importance is Turner's observation that his anterior mem- 
branelle fibril is identical with Yocom's anterior cytostomal fiber, because this 
obviously integrates structurally the neuromotor apparatus described by 
Yocom and the external fibrillar system described by Turner. 

3. The "ventral network," which is entirely comparable, in its general 
features, with the dorsal network, and includes also primary and secondary 
longitudinal fibrils and rosettes, with their central bristles. 



208 CILIATE FIBRILLAR SYSTEMS 

Its pattern, although "constant and characteristic," is less regular and 
"reminds one of badly treated chicken wire." 

Sectioned material showed the fibrils of both dorsal and ventral networks 
"to be immediately under the pellicle and in contact with it." 

The fibrils stained intra vitam were distinctly more delicate than those 
impregnated with the silver. 

Turner confirmed Yocom's observations on the neuronmotor apparatus, 
excepting the motorium. In the E. patella which he studied, he was 
unable to detect this cited organelle. Instead, the single fiber, formed 
by fusion of the five anal cirri fibrils, was traceable to the "collar," 
without a break, where it continued as the anterior memhranelle fbril 
(Turner's designation) noted above. 

4. Vorticella. — The vorticellids, by their size and quick reactions, 
caught the eye of the earliest microscopists, including, of course, 
Leeuwenhoek. The sudden contraction of the spiraling stalk along with 
the inversion and closure of the adoral membranelles naturally invited 
speculations on the kinds of mechanisms that might account for such 
reactions. Geza Entz (1893) cites Wrisberg (1765) as among the first 
to describe "mit recht trefifenden Worden" this surprising behavior 
and to point out its elastic nature. 

To Ehrenberg (1838), however, apparently should go the credit for 
the earliest detailed studies of the fibrous nature of the contractile stalk 
and the detection of longitudinal and circular fibers in the body of 
several vorticellids. He attributed to all of these fibers a contractile 
function and described in the stalk "muscle" cross striations comparable 
to those of the striated muscles of other animals. 

This fibrillar complex in these peritrichs came to be a favorite object 
of investigation by many able workers, especially during the latter half 
of the past century: Dujardin (1841), Czermak (1853), Lachmann 
(1856), Lieberkiihn (1857), Kuhne (1859), Rouget (1861), Cohn 
(1862), Haeckel (1863), Metschnikoff (1863), Kollicker (1864), 
Greeflf (1871), Everts (1873), Engelmann (1875), Wrzesniowski 
(1877), Forrest (1879), Maupas (1883), Brauer (1885), Butschh 
(1889), Schewiakoff (1889), and Entz (1893). The literature for this 
period has been reviewed by Greefif (1871), Wrzesniowski (1877), and 
Biitschli (1889). Similar investigations on the vorticellids have been 
relatively meager during the present century, and the most detailed and 



CILIATE FIBRILLAR SYSTEMS 



209 



careful analysis of the finer structure of their fibrillar system, so far as 
I have found, is that of G. Entz (1893) on "Die elastischen und con- 
traction Elemente der Vorticellen." In the brief review here presented 
of this system, I have followed chiefly this excellent account. 

In this review of the fibrillar system of Vorticella and its relatives, 




Figure 75. Stalk of Zoothamnium arbuscula. (Entz, 1893.) 
axo. — Axonem cyt. — Cytophane spr. — Spironem sps. — Spasmonem 

it will be more convenient to consider first the fibrils of its contractile 
stalk, then those of its body and its peristome. 

Previous to the critical investigations of Entz (1893), the "Stiel- 
strang" in the contractile stalk of the vorticellids, which had been 
identified by Ehrenberg (1838) as the "Stielmuskel," was found by 
later analysts to be composed of two parts: (1) the "Stielmuskel," a 
cylindrical, or band-like, strongly refractive fiber, and (2) an adjacent, 
granular "Protoplasmastrang," which accompanied the former through- 



210 



CILIATE FIBRILLAR SYSTEMS 



out its course. Both were surrounded by a delicate membrane, the 
"Strangscheide," in the same manner that the whole St iel Strang is en- 
closed within the outside membrane, "Stielscheide," of the stalk. 

The Stielmuskel, so most authors (e.g., Engelmann, 1875; Wrzesniow- 
ski, 1877) agreed, was made up of distinct fibrils running variously 
transverse or parallel to its long axis. For Biitschli (1889), however, 
this composition of the Stielmuskel represented rather (and probably 
more in line with his alveolar hypothesis ) an attenuated meshwork. And 
Ehrenberg (1838), Leydig (1883), et al. could apparently see cross 
striations in the Stielmuskel of some vorticellids, comparable with those 
of metazoan muscle. 

Entz (1893) described for the giant stalk of Zoothamnium arhuscula, 




Figure 76. Spasmonem 
(cross section) in Zoo- 
thamnium. (Entz, 1893.) 



and as typical for all the Contractilia, a Stielstrang comprising three 
well-defined fibers: a "Spasmonem," a "Splronem," and an "Axonem" 
(Fig. 75), all enclosed within the Strangscheide. He identified the 
Spasmonem with the Stielmuskel (noted above), and the Spironem and 
Axonem with the Protoplasmastrang. 

In the smaller branches of Z. arbuscula, the Spasmonem (Stielmuskel) 
is a round fiber which, in the main stalk, becomes compressed by the 
adjacent Spironem so that a cross section of the former appears crescent- 
shaped or, since one edge of the crescent is swollen, rather like the form 
of a comma (Fig. 76). As shown by Engelmann (1875), it is bire- 
fringent and may frequently be tinted a steel-gray or appear faintly 
greenish or yellowish. It is stainable in carmine and disintegrates in 
alkali and in mineral salts — reactions which are more rapid in the 
younger stems, and which, according to Entz, would indicate that the 
Spasmonem is not cellulose; neither is it chitin nor keratin, although 
least unlike chitin. 

This age difference shows itself also in the finer structure of the 



CILIATE FIBRILLAR SYSTEMS 211 

Spasmonetn. In the younger branches it is fairly homogeneous, but in 
the older stems and in the main trunk one may distinctly observe, in 
its hyaline ground substance, parallel longitudinal fibrils that gradually 
disappear toward the distal end. Nodal interruptions in these parallel 
fibrils apparently account for the cross striations cited by earlier workers. 
Outside these longitudinal fibrils is a single, or composite, spiraling 
fibril, and centrally placed along the Spasmonem are ovoid discs, each 
containing a central granule or "nucleus." 

Entz's Spironem and AxonetJi constitute, as noted above, the Proto- 



cir. fib. 
1. fib. 



Ci/tO, z^li=^^^^^ 

Figure 77. Three components of Spironem of Zoothamnium. (Entz, 1893.) 
cir. fib. — circular fibril cyto. — cytophanes 1. fib. — longitudinal fibrils 

plasmastrang. This structural duality had been previously overlooked, 
perhaps because the Spironem is wound closely around the Axonem. 
Also, the spirals of the former, which are contiguous when the stalk 
is fully contracted, separate increasingly as the stalk is extended, so that 
the two fibers may easily appear as one in the completely extended stalk. 

In its finer details, the Spironem (Fig. 77) shows beneath its investing 
membrane a spirally wound fibril, under which are several longitudinal 
fibrils. All these fibrils are birefringent and, as noted for those in the 
Spasmonem, nodes of less refringence give here also the effect of cross 
striations. Along the axis of the Spironetn are oval bodies [Cytophanes) 
containing each a central granule [Caryophane). These Cytophanes are 
connected by a longitudinal fibril (Fig. 75) similar to a string of pearls. 

In the Axonem (Fig. 75) longitudinal fibrils comparable to those in 
the Spironem also occur, but their course is apparently completely inter- 



212 



CILIATE FIBRILLAR SYSTEMS 



mpted at regular intervals by Cytophanes which are relatively much 
larger than those found in the Spironem. 

It seems to be generally agreed that the stalk of all the vorticellids, 
both the Contractilia and the Acontractilia, is a direct continuation of the 
body. Bearing this in mind, we may now briefly review the fibrillar 



pe//. ___ 




i.mc/o. 



Figure 78. Pellicular structure, and branching of longitudinal myonemes in Zootham- 
nium. (Entz, 1893.) 
Ion. myo. — longitudinal myoneme pell. — pellicle 

system of the body proper and later note how the parts of this system 
are related to those in the stalk. 

For convenience in description and with special reference to the genus 
Vorticella, we may regard the body as divisible into three fairly well- 
defined regions : ( 1 ) the funnel, lying between the stalk and the ciliary 
ring; (2) the bell, that part of the body above the ciliary ring; and 
(3) the disk, which includes the peristomal border, adoral zone, and 
cytostome. 

The entire body, like the stalk, is covered by a pellicle which, accord- 



CILIATE FIBRILLAR SYSTEMS 213 

ing to Entz (1893), is not homogeneous but distinctly sculptured (Fig. 
78) as if composed of "Stabchen" that overlap, somewhat as tile on 

a roof. 

The fibrillar system of the body lies immediately beneath the pellicle 
and comprises an outer and an inner complex of fibrils, or myonemes. 
Each such complex is in turn composed of ( 1 ) an outer circular layer, 
and (2) an inner longitudinal layer, making in all, then, four fibrillar 

layers. 

Lachmann (1856) was first to describe the outermost circular layer. 



Ion. my 

o.sp.mt/' 




Figure 79. Myonemes of stalk sheath of Vorticella. (Entz, 1893.) 
Ion. my. — longitudinal myoneme o. sp. my. — outer spiral myoneme 

It was later recognized also by Stein (1867), but apparently overlooked 
by other investigators previous to Entz (1893). As a single fibril, it 
spirals directly beneath the pellicle and may be followed from the 
attachment of the stalk uninterruptedly to the center of the disc. Its 
spiral course accounts for the annular appearance of the pellicle. Entz 
(1893) thinks also that the birefringence of the pellicle may be due 
to this underlying fibril, since, as shown by Engelmann (1875), all the 
fibrils are refractive in polarized light. 

The fibrils of the next layer, the longitudinal fibrils, lie immediately 
below layer (1), noted above, and likewise pass from the style's attach- 
ment to the center of the peristomal disc. On this disc they of course 
are radially arranged. This layer was found by Greeff (1871), but 
Bijtschli (1889) questioned its existence. 

It should further be noted also that this outer fibrillar complex is 
continued uninterruptedly into the protoplasmic lining of the style sheath 
(Fig. 79). This will be referred to again in later discussion. 



214 



CILIATE FIBRILLAR SYSTEMS 




pQr. bdr. 



mer.mi 



yo. 



fnl. msc. 



Figure 80. Arrangement of second complex of body fibrils in Ephtylis. (Entz, 1893.) 
fnl. msc- — funnel muscle mer. myo.^ — meridional myoneme 

per. bdr. — peristome border 

Of the second complex of fibrils, its outer component, unlike the outer 
single fibril of the first complex, behaves rather as a large fiber that may 
become split into several fibrils. This set of fibrils courses spirally around 
only the lower half of the funnel portion of the body (Fig. 80) . Toward 
the ciliary ring it disappears and, according to Entz (1893), reappears 
actually to form this ciliary ring "out of a number of fine fibrils (Myone- 



CILIATE FIBRILLAR SYSTEMS 215 

men)." This would account for the interlaced appearance of the ciliary 
ring. Above the ring, in the bell part of the body, this set of fibrils could 
not be detected except in the peristome border, where it forms a fairly 
strong spiral, narrowly wound. Its occurrence there was described by 
earlier authors as a "sphincter ring" of the peristome border (Fig. 80). 

On the disk, this fiber appears as a fairly thick strand, one end of 
which originates in the center of the disk and, after following at their 
base the several turns of the adoral membranelles along the outer margin 
of the disk and through the mouth into the gullet, it continues down the 
inner surface of the gullet the whole length. Along the gullet wall Entz 
thought that, in some preparations, he could observe this fiber branching 
into many increasingly fine fibrils. 

The inner, longitudinal layer of the second fibrillar complex is a 
direct continuation of the Spasmonem (Stielmuskel) of the contractile 
stalk. Upon entering the body this Spasmonem breaks up into its com- 
ponent fibrils, which diverge and so form the "funnel muscle" (Fig. 80) 
of earlier authors. Higher in the funnel, these fibrils may rebranch, then 
anastomose into a network. At the ciliary ring, the fibrils curve round 
it as crescent-like spans, which are somewhat larger and apparently more 
dense, and continue directly up the wall of the bell to the peristome 
border. These meridional myonemes (Fig. 80) may end singly on this 
border or, as Engelmann (1875) noted, they may bifurcate and unite, 
each of a pair with its adjacent neighbor, to form "arcades" just below 
the peristome border. The fibrils then span this projected border, much 
as they curved over the ciliary ring, and thereupon proceed as radii to 
the center of the disc. Toward this center, these fibrils may sometimes 
branch and anastomose to form varied and striking patterns. 

Finally, it may be mentioned that, in addition to the four layers of 
fibrils noted above, Entz (1893) describes a fairly thick strand of other 
fibrils which originate from the middle of the disc and project down- 
ward through the endoplasm and finally disappear in the region of the 
cytostome. 

B, INTERPRETATION 

The essential structural components of the fibrillar systems of several 
representative ciliates have been briefly reviewed in the foregoing para- 
graphs. It remains now to consider the several functions that have been 



216 CILIATE FIBRILLAR SYSTEMS 

ascribed in the literature to each of these fibrillar mechanisms, as illustra- 
tive for similar systems that have been studied thus far in a large number 
and variety of other ciliates. 

It will be recalled that Ehrenberg (1838) saw in the "stalk muscle" 
of Vortkella cross striations which he regarded as comparable to those 
of the striated muscles of other animals. Also, he detected both longi- 
tudinal and circular fibers in the body of several other vorticellids. Quite 
in line with his search for such comparisons, he attributed to all of these 
fibrils a contractile function. For him they were literally miniature 
muscles which had their structural and functional counterparts in the 
muscles of macroscopic organisms (Maupas, 1883). Evidently, Ehren- 
berg's comparison between microorganisms and macroorganisms led him 
to look for a one-to-one correspondence between microorgans and 
macroorgans. 

It will be recalled that what Ehrenberg regarded as the stalk muscle 
(Stielmuskel) was analyzed into several components by later investi- 
gators. These, according to Entz (1893), included: a cross-striated, rod 
or band-like Spasmonem, and another rod-like strand, the Axonem, 
around which was coiled the Sphonem. Each of these was, in turn, 
analyzable into longitudinal fibrils, whose properties apparently ac- 
counted for their birefringence and cross striations. The Spasmonem and 
Spironem showed also, spiraling within their own spiral strand just 
outside its longitudinal fibrils, a fine fibrillar coil coursing throughout 
their length. Also, both Spironetn and Axonem had, centrally located, 
the "cytophanes" which, with their longitudinal interconnections, ap- 
peared like a string of pearls. Thus Ehrenberg's "Stielmuskel" turns out 
to be a highly complex mechanism which is structurally quite different 
from the "muscles of other animals" to which he had compared it. 

Finally, mention was made also of the circular and longitudinal 
myonemes of the protoplasmic lining of the style sheath, which are 
continuations of the outer fibrillar complex of the body myonemes. 

Taking into account this assemblage of highly differentiated com- 
ponents within the stalk of the Contractilia, together with a few rather 
inconclusive experimental results, various interpretations were advanced 
to account for both the contraction and the extension of this stalk. These 
may be summarized as follows: 

1. The majority of investigators agree that the quick spiraling con- 



CILIATE FIBRILLAR SYSTEMS 217 

traction of the stalk is referable to contractile properties of some one 
or more of its components. Dujardin (1841) was apparently the only 
one to regard the stalk sheath as the seat of that contractility. Ehren- 
berg's Stielniuskel was for him the contractile component, but also the 
only component that he had recognized within the stalk sheath. Of the 
three fibers into which this StieUnuskel was later analyzed, viz., the 
Spasmonem and Spironem and Axoyieni, Kiihne (1859) considered 
the latter two, or Protoplasmastrang, as the contractile organelle. This 
he likened to the sarcoglia of metazoan muscle. Other proponents of 
this contractile theory (Czermak, 1853; Engelmann, 1875; Wrzesniow- 
ski, 1877; and others) regarded the Spasmonem as the contractile fiber 
which, for later authors, represented a bundle of fibrils or myonemes 
that continued without interruption into the body of the vorticellid. 

2. Apparently there is general agreement among all the investigators 
that the mechanism of stalk extension inheres in the elastic properties 
of the pellicle (Butschli, 1889; Entz, 1893). Only Kuhne (1859), who 
alone regarded the Spironem and Axonem as the contractile organelles, 
attributed elasticity to both the pellicle and the Spasmonem, thus to 
account for the stalk's extension. 

3. While the majority, as noted, support the contractile theory of 
stalk contraction, there is a minority group who maintained that the 
vorticellid stalk, in its quick spiraling retraction, actually does not con- 
tract as such but instead recoils somewhat as a coiled spring. 

A chief proponent of this elastic theory is Entz (1893), who sees the 
Spasmonetn not as a contractile fiber but, like Kiihne, as an elastic fiber. 
This organelle is, according to Entz, primarily responsible for the sudden 
recoil of an extended stalk. He likens this elastic fiber to a curly hair 
which when stretched and then released will resume its spiral form. 

The opposing force, tending to "stretch" this normally spiral Spas- 
monem, is inherent in the elastic pellicle. Thus pellicle and Spasmonem 
constitute a pair of "antagonistic elements." 

Associated with this pair is another pair of antagonistic elements, 
viz., (1) the longitudinal myonemes in (a) the stalk sheath and (b) 
the Spironem; and (2) the circular myoneme in (a) the stalk sheath 
and (b) the Spironem. These opposing pairs of fibrils are relatively 
weak, but of a strength sufficient to determine by their antagonistic 
contractions whether the stalk "contracts" with the recoil of the elastic 



218 CILIATE FIBRILLAR SYSTEMS 

Spasf7ionem or extends by means of the elastic pellicle. Accordingly the 
longitudinal fibrils of the stalk sheath and the Spironem tend to reinforce 
the elasticity of the Spasmonem, and the spiral myoneme of both sheath 
and Spironem, upon relaxation of the longitudinal pair of myonemes, 
tend to reinforce the elasticity of the pellicle. 

Entz's rather elaborate thesis at least makes evident the elaborate 
differentiation of the vorticellid's stalk, and so provides one working 
hypothesis which might be experimentally tested. With modern tech- 
niques, this should not prove very difficult. And, in the writer's opinion, 
the vorticellids offer extraordinary possibilities for some clear-cut and 
fruitful experiments which should help toward a better understanding 
of their fibrillar mechanisms. 

It seems to be more generally agreed that the fibrillar complexes of the 
funnel, bell, and disc are primarily contractile. If so, and if the 
Spasmonem of the stalk is, as Entz and others claim, continued without 
interruption into the funnel and bell as the longitudinal fibrils of the 
"inner complex," then it should follow that if these fibrils of the inner 
complex are contractile, one should expect the Spasmonem also to be 
contractile. Entz (1893) apparently does not discuss this point. 

Finally, it may be noted that Entz's thesis attributes no specific function 
to the Axonem. Both he (1893) and Stein (1854) suggest that it might 
function as a neuroneme. 

Observations on the fibrillar system of Stentor have provided a basis 
for some interpretations which appear more plausible than those cited 
above for the vorticellids. These interpretations have to do exclusively 
with the myonemes. The other fibrils are poorly understood. 

Before discussing these, however, the essential components of this 
system should be recalled to mind. As was pointed out, and as will 
doubtless be familiar to the reader, the myonemes of Stentor are band- 
like fibers which course beneath and slightly lateral to the longitudinal 
rows of body cilia and the curved ciliary rows on the oral disc. 

Associated with these band-like myonemes, but lying peripheral to 
and mostly contiguous to them, are other fibrils which Neresheimer 
(1903) described as "neurophanes" and which Dierks (1926) recently 
further described and named "neuroids," since he could not fully identify 
these with Neresheimer's neuronemes. 

Dierks added one very significant observation, which, if verifiable. 



CILIATE FIBRILLAR SYSTEMS 219 

would definitely suggest a conductive function for his neuroids. He saw, 
in several preparations, that the smaller fibril gave off one or more 
branches to its adjacent myoneme. As will be noted further on, this claim 
has recently been challenged by von Gelei (1929b). 

Another fibril, found and described by Schuberg (1890), connected 
the basal lamellae of the entire series of membranelles. This basal fibril 
was noted also by Johnson (1893), Maier (1903), and Schroder 
(1906), but Neresheimer and Dierks maintain that such does not exist. 
Dierks (1926) does describe a fibril coursing along the platelets of the 
membranelles, which he thinks both Schuberg (1890) and Meyer 
(1920) may have seen but misinterpreted. 

The evidence supporting the interpretation that the myonemes are 
contractile organelles has been supplied from many sources, beginning 
with their discoverer, Lieberkiihn (1857). Ehrenberg (1838) claimed 
to have seen in a living Stentor that in the extended state the myonemes 
were serpentine, while with the contraction of the body they became 
shortened and straight. But since he misidentified the pigmented meridi- 
ans as myonemes, the significance of his observation is uncertain. 

Probably the most significant evidence for the contractile nature of 
Stentor' 5 myonemes was provided by Johnson (1893) and verified by 
Dierks (1926) . The former investigator compressed the living organism 
beneath the cover slip and could then observe the myonemes "alter- 
nately to extend and contract," concluding that "no one who has once 
observed them under these conditions can doubt that they are responsible 
for the contractions of the animals." Biitschli (1889) apparently made 
similar observations on living Stentor upon applying an electric stimulus. 
Merton (1932) has confirmed these former evidences of the contractile 
properties of Stentor' s myonemes. 

Dierks (1926) states further that "these fibrils are definitely shorter 
and thicker in the contracted animal and longer and shorter" when the 
organism is extended. He also noted, however, that this behavior was 
lacking in the myonemes toward the posterior end of the body, and 
supposed that here the body protoplasm contracted more or less inde- 
pendently, suggesting a progressive differentiation in the myonemes 
anteriorly along their course. 

The assigned contractile function of the myonemes of Stentor appears 
to be agreed upon without exception. Schroder (1906) added to the 



220 CILIATE FIBRILLAR SYSTEMS 

knowledge of their contractile function probably also that of conducting. 
But for the other fibrils of this ciliate, evidence regarding their actual 
function or functions is mostly wanting. Interpretations based on mor- 
phological evidence concern chiefly the relations of these fibrils (neuro- 
phanes or neuroids) to other organelles. 

Neresheimer's observation on the parallel course of the neurophanes 
with the myonemes and the differential stainability of these two kinds of 
fibrils, suggested for the neurophanes a conductive function. By means 
of a drop-weight apparatus (Fallmachine) he tested the responses of 
Stentor to a variety of narcotics: morphine, strychnine, atropine, caffein, 
and so forth, and to other chemicals which are known to affect the nerves 
of higher animals. On the basis of an assumed selective action of these 
reagents, he derived evidence which seemed to favor assigning a nervous 
function to the neurophanes. The technique for these tests was ingeni- 
ously devised and might prove useful also for others. But owing to lack 
of adequate controls and because of other possible interpretations, his 
results do not seem very convincing. 

Schroder (I906) considered Neresheimer's interpretation of the 
neurophanes invalid. He believed that Neresheimer had mistaken a struc- 
tural feature of the "mid-stripes" for his neurophanes, as indicated upon 
comparing the latter's Figures 7 and 8 with Schroder's Figures 1-5. 

From Dierks' more extensive and detailed studies, however, there can 
be no doubt about the identity of another fibril coursing with each of 
the myonemes. The occurrence of these was consistent and their relation- 
ship with the myonemes apparently significant. As formerly noted, 
Dierks found that in good preparations branches were given off from 
the neuroids to the myonemes which they evidently joined. He considered 
several possible functions which this connection between neuroids and 
myonemes might indicate, and finally, as a plausible hypothesis, sug- 
gested for them a conductive function. 

Von Gelei (1926c) rejects this interpretation. From his own previous 
studies (1925) on Stentor, he concludes that Neresheimer's neurophanes 
and Dierks's neuroids are real and are identical structures. But, according 
to von Gelei, these neuroids are not fibers but bands composed of fibrils, 
which are quite wide, especially in the aboral region. They are not a 
structural feature of the pellicle (Schroder, 1906), but are sub pellicular 
and fused to the pellicle. This was indicated in contracted Stentors by 



CILIATE FIBRILLAR SYSTEMS 221 

the body groove overlying the neuroid, as produced by the contraction of 
the myoneme under the neuroid. The attachment of the pelHcle to the 
neuroid and the latter, in turn, to the myoneme would, upon contraction 
of the myoneme, cause an ingrooving of the pellicle. 

Studies on the hypotrichous ciliates yielded apparently the first re- 
corded example of fibrils directly associated with ciliary locomotor 
organelles. Engelmann (1880) traced such fibrils from the bases of the 
marginal cirri of Stylonychia "nach der Mittellinie des Leibes." He 
postulated for these fibrils a conductive function in transmitting im- 
pulses, as in nerves of higher animals, from the ventral region of this 
hypotrich to the cirri, the movements of which might thereby be 
regulated. 

Engelmann's interpretation was formulated entirely by analogy and 
he offered no substantial evidence to support it. It seems to have been 
generally rejected by his contemporaries. Maupas, who three years later 
(1883) described similar fibrils in E. patella var., said, in a brief foot- 
note (p. 622) concerning his own findings: "Quant a la signification 
physiologique de ces racines ciliaires, j'avoue ne pas la connaitre." And 
with reference to Engelmann's discovery of "fibrilles nerveuses" in 
Stylonychia myttlus, Maupas adds: "Je ne sais comment concilier des 
interpretations aussi divergents." Biitschli regarded Engelmann's inter- 
pretation as untenable and proposed, on equally meager evidence, a 
contractile function for these fibrils. Maier (1903), who attributed a 
contractile function to the basal fibril associated with the basal lamellae 
of membranelles in Stentor, concluded that the lateral cirri fibers in 
Stylonychia were "required" for the support of these cirri. 

Prowazek (1903), on the other hand, suggested that the anal cirri 
fibers which he found in E. harpa might perform the dual functions of 
contractility and conductivity. He noted, however, that E. harpa could 
move each anal cirrus independently and could flex the tip of these cirri 
quite at will. He observed, moreover, that a detached anal cirrus might 
continue its contractions for a time. These important observations have 
apparently been overlooked by later investigators. 

It will be recalled that Grifl&n (1910) compared the anal cirri fibrils 
of E. ivorcesteri, which he found and described, with myonemes whose 
number might have become reduced phylogenetically during reduction 
in the hypotrichs's rows of cilia. He pointed out a difficulty in this 



222 CILIATE FIBRILLAR SYSTEMS 

interpretation, viz., that some of the few fibrils of other cirri in this 
species were not ahgned longitudinally, but might be even transverse to 
the longitudinal axis. 

Griffin based his concept of contractility for these fibrils in E. Worces- 
ter/ only partly, however, on this comparison with myonemes in other 
ciliates. He accepted Biitschli's interpretation of a contractile function 
for the marginal and anal cirri fibrils in Stylonychia as a much more 
reasonable view, and also noted that "Every detail of arrangement and 
structure indicates that the fibrils are, principally at least, contractile in 
function." He observed, also, that the fibrils were developed around the 
bases of the cirri in such a way as to assist in producing the ordinary 
motions. "As the anal cirri have only a single strong motion, a vigorous 
kick directed backward, each needs but a single strong fibril." 

Evidently Griffin had not seen any reversal in the effective stroke of 
these anal cirri. In several other species of Euplotes this reversal is not 
uncommon in both swimming and creeping movements. Should that be 
the case in E. tvorcesteri, then a contractile function for the anal cirri 
fibers is scarcely conceivable. The effective function of a contractile fibril 
is obviously a pulling but not a pushing function. Moreover, Prowazek's 
account of the contractile behavior of the anal cirri in E. harpa, cited 
above, largely vitiates any claims for contractility in the cirri fibrils of 
this species, and similar behavior has been observed by the writer in the 
anal cirri of E. patella. It was also noted (Taylor, 1920) that cutting 
these fibrils apparently did not impair the effective stroke of the anal 
cirri, whether that stroke was directed backward or forward. 

The latter evidences were thus cited against the assumption that these 
fibrils in E. patella were contractile. It was further shown by those 
experiments that cutting the anal cirri fibrils interrupted the coordinated 
movements between anal cirri and adoral membranelles. Also, severing 
the membranelle fiber likewise interrupted the coordinated movements 
of the membranelles on opposite sides of the incision. Incisions in other 
parts of the body did not impair the coordination of these organelles. 
It should be pointed out, however, that those incisions which did inter- 
rupt coordinated movement of organelles cut not only the anal cirri 
fibrils or the membranelle fiber, but also the peripheral fibrils which 
have since been described especially by Turner (1933). Whatever role, 
if any, these peripheral fibrils may have in E. patella's coordinated 



CILIATE FIBRILLAR SYSTEMS 223 

behavior was not demonstrated by the writer's (1920) experiments. 
Reinvestigation of this problem, especially on a more favorable form 
such as Lkhnophora (Stevens, 1891), ought, therefore, to be undertaken 
in order to determine what relative roles the so-called introplasmic fibrils 
and the peripheral fibrils each perform in the coordinated movements 
of the organelles with which such fibrils are demonstrably associated. 

Several investigators (Belar, 1921; Jacobson, 1931; Peschowsky, 
1927) have maintained that all such cirri fibrils in the hypotrichous 
ciliates and the fibrillar systems in various other ciliates are primarily 
or exclusively supporting in function. Jacobson (1931), for example, 
studied by means of various techniques, including the silver-nitrate 
methods, the fibrillar systems of some twenty-seven ciliates. These com- 
prised representatives of all the major groups and included among the 
hypotrichs £. patella and £. char on. She concluded from the results that 
no evidence was found in support of a conductive function for any of 
the fibrillar systems studied. It was pointed out that in the hypotrichs 
whose motor organelles are localized, fibrils are nevertheless present, 
as on the dorsal side where cilia are wanting. Reference on this point 
should be made to Turner's (1933) studies on £. patella, which showed, 
as previously mentioned, that longitudinal fibers connect the bases of 
the dorsal and ventral rows of bristles, whose function is not known. 
But they may have a function and if that function is, as has been 
suggested, sensory in nature, it is surely not inconceivable that the 
associated fibrils may facilitate its performance. 

Another and more significant observation was made by Jacobson, viz., 
that in Sciadostoma difficile, where three ciliary rows surround the 
anterior body pole, no silverline connection exists between the basal 
granules. An impulse, therefore, originating at the anterior ciliary ring 
would need to pass nearly to the posterior body pole and back again in 
order to affect adjacent cilia. Since our assumptions should be, first of 
all, plausible, one would justifiably regard this morphological evidence 
contradictory to the thesis that these silverlines in 5". difficile are con- 
ductive in function. It may be pointed out, however, that Chatton and 
Lwoff (1935) apparently demonstrated that the fibrils (cinetodesme) 
described for several holotrichous cihates, which alone were connected 
with the basal granules, could not be stained by silver impregnation, 
whereas other adjacent fibrils (interpreted by them as silverline fibrils) 



224 CILIATE FIBRILLAR SYSTEMS 

could be silver impregnated but apparently were not in contact with the 
basal granules. 

The thesis that ail fibrils of all ciliates are only supportive in function 
is, of course, not tenable. One would at once except myonemes. But why 
limit the exceptions to myonemes.'' If, in the eons of time, protoplasmic 
fibrils have become difi^erentiated so as to facilitate contractility in 
protistan organisms, who can deny them the capacity to have become 
differentiated also to facilitate conductivity or some other function in 
these unicellular forms of life? All our assumptions should be both 
plausible and reasonable assumptions, the validity of which may, in the 
last analysis, be demonstrated only by experiment. 

In conformity with the less specialized differentiation of its motor 
organelles, the fibrillar system of Paramecium is also relatively less 
specialized, as compared with those of the other three representative 
ciliates reviewed in foregoing paragraphs. 

From the accounts especially of Schuberg (1905), von Gelei (1925- 
31), and Lund (1933), it appears that two separate and distinct com- 
plexes have been described in the literature, which may be represented 
by the outer fibrillar complex of von Gelei and of Klein (1926-32), 
and by their inner fibrillar complex. For these latter authors, both com- 
plexes are subpelUcular. 

According to Schuberg (1905) and Lund (1933), however, the 
above-mentioned outer fibrillar complex is not subpellicular, but actually 
represents the polygonal pattern of the pellicle itself. Lund emphasizes 
the fact, therefore, that the essential fibrillar system of Paratnecium is 
exclusively the complex which is associated with the basal granules of 
the entire motor mechanism, including the cilia of the mouth, cyto- 
pharynx, and cytoesophagus, as well as the body cilia. Lund's fibrillar 
system would essentially include, therefore, the inner fibrillar complex 
of von Gelei and of Klein. 

The discrepancies just noted in the structural interpretations of 
Paramecium's fibrillar system are obviously crucial, since they go hand 
in hand with discrepancies in the functional interpretations of that 
system. This holds, of course, not only for the investigators cited above, 
but for various others also. 

Referring now to these diverse functional interpretations, Schuberg 
(1905) suggested that the fibrils connecting the basal granules, such as 



CILIATE FIBRILLAR SYSTEMS 225 

he had described for Paramecium and Frontonia, might function in the 
well known metachronism of ciliary movement. He was not inclined, 
however, to compare these fibrils with Neresheimer's neurophanes nor 
with the neurofibrils of the Metazoa. Obviously, since it had not yet 
been proved that the neurofibrils were the conductive elements of nerves, 
then an analogy between these neurofibrils and the fibrils found in 
Paramecium would not add to our understanding of either the one or 
the other. 

Mention may here be made of the system of fibrils described for 
Paramecium by Rees (1922). These have an arrangement and relation- 
ship quite different from any of the fibrils referred to above. According 
to Rees, all of the fibrils he observed connected the basal granules of 
the cilia of the body and cytopharynx with the motorium, located just 
anterior to the cytostome, by coursing through the cytoplasm in several 
graceful whorls. 

From the results of his few experiments, Rees concluded that these 
fibrils were conductive in function. By severing with a microneedle the 
fibrils connecting the cytopharyngeal membranelles with the motorium, 
the coordinated movements of these membranelles was interrupted. Like- 
wise, the coordinated movement of body cilia was interrupted when the 
neuromotor center was destroyed. 

In view of the later descriptions of the more peripheral system of 
fibrils in Paramecium, Rees's experiments should be repeated. It may be 
noted also that Jacobson (1931) reinvestigated this fibrillar complex 
described by Rees, and concluded that he had observed not fibrils but 
internally discharged trichocysts, as effected by the killing agents used. 
Her figures of fixed Paramecium illustrating such trichocysts are com- 
parable to some of Rees's figures, except for certain regularities in the 
"fibrillar whorls" depicted by Rees. One could suppose that the in- 
ternally discharging trichocysts might tend to follow the course of these 
whorls of fibrils and so reproduce that course in fixed material, which, 
through differential staining, revealed the trichocysts but not the fibrils. 
Only further careful investigation can clarify this discrepancy. 

The interpretations of von Gelei (1925-31) and of Klein (1926-32) 
agree in ascribing a conductive function to the inner fibrillar complex 
of Paramecium, but their views on the relations and functions of its 
outer fibrillar complex are not in accord. The basis for their interpreta- 



226 CILIATE FIBRILLAR SYSTEMS 

tions is confined primarily to morphological evidence. This evidence is 
derived, however, not only from their studies on the fibrillar system of 
Paramecium, but also from comparative studies of a considerable number 
and variety of other ciliates. 

According to von Gelei's interpretation, Klein's strongly birefringent 
"indirekt System" (Meridian II. Ordnung) is nothing more than the 
well known "pelliculare Gittersystem" of the ciliates, which functions 
as a supporting network to maintain body form. As such, it has no 
differentiated structural connections with the less refractive inner fibrillar 
complex (von Gelei's neuroneme system), which alone, therefore, is 
the peripheral conductive mechanism. Von Gelei sees in the longitudinal 
and transverse fibrils of his neuroneme network a mechanism which 
structurally integrates the ciliary basal granules, the trichocysts, and even 
the contractile vacuole pore into a coordinated whole ("einer koordi- 
nierten Einheit"). 

This integrated mechanism coordinates automatically the metachronous 
effective strokes of successive cilia. The direction of the stroke of one 
cilium activates conditionally that of the next by way of the basal 
apparatus (basal granule, basal ring, and "Nebenkorn"), which repre- 
sents a sensory organelle. For the coordinated activity of the organism 
as a whole, however, von Gelei recognizes in his peripheral neuroneme 
system the absence of a neuromotor center. Accordingly, he regards the 
"intraplasmatic" fibrillar complex described by Rees as a centralized 
mechanism which may complement the peripheral neuroneme complex, 
thus to provide reasonably a unified neuromotor system. Von Gelei 
(1929b) observed by means of his silver-osmium-formol method a 
"platte" beneath the basal apparatus, which, he thought, might serve 
as a sort of end plate connecting Rees's intraplasmic fibrils with the 
peripheral neuroneme complex. 

As has been previously indicated, Klein's (1926-32) interpretation 
of his inner fibrillar complex (Meridian I. Ordnung) agrees essentially 
with von Gelei's concept of a conductive function for his peripheral 
neuroneme network. Among their minor differences, Klein would ac- 
count for the commonly observed reversal in the effective stroke of cilia, 
not by means of Rees's intraplasmic complex as von Gelei supposed, but 
by way of a "primitive reflex arc." This reflex arc includes the axial 
filament of the cilium as the receptor, the basal apparatus as the ""relator," 



CILIATE FIBRILLAR SYSTEMS 227 

and the protoplasmic sheath as the effector. The basal apparatus com- 
prises the basal granule and two "Nebenkorner." The latter serve to 
spread the proximal part of the reflex arc and, with the basal granule, 
may function as a kind of commutator that regulates the direction and 
change in direction of the effective stroke of the cilium. 

We may note that while such a mechanism might conceivably account 
for a reversal in the effective stroke of a given cilium, obviously it does 
not, as such, provide for the synchronous reversal of the many other 
cilia, which is an essential part of the problem of coordinated movement. 

In this connection, reference may be made to a fairly recent paper 
by J. C. Hammond (1935), who would refer the phenomenon of 
synchronous and metachronous ciliary behavior to an anterior-posterior 
physiological dominance in the organization of the cell, as opposed to 
the concept of a neuromotor m'echanism. This thesis might help to 
account for this more general coordinated behavior (Rees, 1921), but 
it would presuppose a reversal in anterior-posterior dominance of the 
physiological axis in order to explain the reversal of ciliary stroke so 
common in the swimming or creeping behavior of ciliates. Moreover, 
the well known localized reversal of a few or many of the cytostomal 
membranelles in the intake or ejection of solids would obviously require 
a similar presupposition. 

The chief discrepancy between Klein's and von Gelei's interpretations 
of the fibrillar system of Paramecium concerns the structural and func- 
tional relations between their outer fibrillar complex and their inner 
fibrillar complex. Klein (1928, p. 203) regards these two fibrillar com- 
plexes as a continuum. His "Meridian II. Ordnung" is a derivative of 
his "Meridian I. Ordnung," as are also all cilia, basal granules, "Neben- 
k5rner," trichocyst granules, and protrichocyst granules (secretory, or 
"Tektin" granules). In its re-genesis, observed during reorganization, 
many more fibrils (Profibrille) are formed than are retained, varying 
with the species and genus, and of those that persist some may unite to 
form composite fibrils (Biindelfibrillen), as occurs in Paramecium 
(Klein, 1932). 

Klein's silverline system incorporates as a unit, therefore, both struc- 
turally and functionally, the outer and inner fibrillar complexes. In its 
re-genesis, it functions as a "form-building system," and some of its 
fibrils become more resistant and rigid by the addition of a secondary 



228 CILIATE FIBRILLAR SYSTEMS 

substance, the "fibrillare Komponente" (1928, p. 255). By virtue of its 
integral relation with the basal apparatus of the cilia and with the tricho- 
cysts, he attributed to his silverline system also a specifically conductive, 
coordinative function. 

Chatton and Lwoff's (1935-36) criticisms of Klein's interpretations 
bear mainly upon the structural relations of the fibril to which are ad- 
joined, always on its left side, the basal granules. The former authors 
think Klein's silverline system is quite separate and distinct from their 
infraciliature. It appears to the writer, however, that this discrepancy 
might be completely resolved by identifying Chatton and Lwoff's infra- 
ciliature with the longitudinal fibrils and basal granules of Klein and 
von Gelei's inner fibrillar complex. Further critical investigation would 
be needed, of course, to establish such identity. 

Fibrillar Systems of Other Ciliates 
a. holotricha 

Ancistruma (Kidder, 1933). — The fibrils in Ancistruma mytili and 
A. isseli are of three types — the longitudinal fibrils of the ciliary rows, 
the irregularly distributed transverse fibrils, and the net complex of fine 
fibrils in the peristomal region. The latter fibrils seem to connect di- 
rectly with the basal bodies of the peristomal cilia, and they are coarser 
and less numerous in A. isseli than in A. mytili. The longitudinal fibrils 
in A. mytili are continuous around the posterior end, but in A. isseli 
they center about a posterior suture. 

A number of fine fibrils connect the inner dorsal row of peristomal 
cilia to the outer dorsal row. These seem to be distinct from the net 
complex and probably are in the nature of concentrated transverse 
fibrils. In A. isseli such fibrils are absent, and instead the fibril of the 
outer ciliary row joins that of the inner dorsal row. 

Fibrils resembling the interstrial fibrils of Boveria (Pickard, 1927) 
are sometimes seen in A. mytili, but these may represent a deep-lying 
network of the same type regularly seen in the peristomal region. 

Methods 

Fixatives: Schaudinn, sublimate-acetic, Bouin's, Zenker's, Champy's. 
Stains: Heidenhain's and Delafield's haematoxylin, crystal violet-sulph- 
alizarinate (Benda's). 



CILIATE FIBRILLAR SYSTEMS 229 

Klein's silver method, modified to include fixation with osmic acid fumes 
and impregnation period of one to three hours in 2-percent solution of 
silver nitrate. 

Boveria teredinidi Nelson (Pickard, 1927). — Surrounding the cyto- 
stome is an oral ring which begins and ends in the motorium. From the 
motorium arise the anterior and the posterior adoral fibrils, which bound 
the adoral zone. The posterior fibril joins the anterior fibril distally, 
and the latter continues as the pharyngeal fibril. This enters the endo- 
plasm in the region of the pharynx and spirals around the potential gul- 
let. A fibril from a point on the ring opposite the motorium enters the 
pharyngeal fibril near the margin of the peristome. Within the peristomal 
field delicate fibrils connect the anterior adoral fibril with the ring. 

The longitudinal lines of the body surface consist of contractile Jiiyo- 
nemes and basal granules of the ciliary rows. Myonemes arise at the 
anterior end directly from the posterior adoral fiber, or indirectly from 
fibrils of the posterior granular line. The myonemes pass posteriorly and 
in somewhat oblique parallel lines. They gradually converge in the pos- 
terior field. Basal granules of cilia rest on tnyonemes, oral ring, and the 
anterior and posterior adoral fibrils (except the free end of anterior 
adoral fibril) . A deep nerve net, consisting of longitudinal interstrial and 
transverse fibrils, interconnect the area "between the myonemes and 
their basal granules." 

Methods 

Fixatives: Schaudinn's (60° C), Bouin, Zenker, formalin, osmic acid, 

Da Fano. 
Stains: Delafield's iron haematoxylin, Mallory's triple, alum carmine, and 

Yabroff's silver-gold method. 
Clearing: Xylol, oil of cedar; equal parts of bergamot, oil cedar, and 

phenol, and sometimes before imbedding, in synthetic oil of wintergreen. 

Chlamydodon sp. nov. (MacDougall, 1928). — The structural analysis 
of this new species of Chlamydodon disclosed "a complex neuromotor 
apparatus, including a coordinating center, and systems of fibers con- 
nected with cilia, the mouth opening, the pharyngeal basket, and the 
'railroad track.' " 

The motorium was identified as a bilobed mass located just below the 
anterior end of the large pharyngeal basket. This mass and all the 



230 CILIATE FIBRILLAR SYSTEMS 

fibrils of the neuromotor system stained bright red by Mallory's method. 
When dislodged along with the basket by means of microdissection, the 
motorium was "a refringent body." In stained sections, however, it ap- 
peared granular. 

"Fans of fibrils" join the motorium at both its anterior and posterior 
ends. Those at the anterior end mark the convergence of longitudinal 
fibrils (Fig. 81 A) of the body cilia, of ventral fibrils from the posterior 
region of the body and of fibrils from the mouth. The circular myonemes 
of the mouth are traversed by many fine fibrils that continue on toward 
the "railroad track." A fibrillar fan from the posterior end of the moto- 
rium continues as the dorsal fibers to the "railroad track," which, in turn, 
show a very complex system of fibrils associated with its trichites. 

The basal bodies of the cilia are connected not only by the longitudinal 
fibrils but also by cross fibrils (Fig. 8 IB). 

By methods of microdissection it was shown that (1) after destruc- 
tion of the motorium "there is a marked disturbance in the action of the 
cilia, in no way comparable to the disturbance of the cilia if other parts 
of the body are injured"; (2) the cilia may still exhibit a wave-like 
motion, but they do not reverse after the motorium is destroyed. This 
seemed to suggest a coordinating function for the fibrillar system of this 
Chlamydodon, whose inconspicuous motor organelles are not favorable 
for an experimental study of modifications of their coordinated activity. 

Methods 

Fixatives: Schaudinn's, Bouin's, and strong Flemming's. 

Stains: Iron-haematoxylin, Mallory's (after Zenker's or picromercuric 

fixation). 
Microdissection. 

Conchophthirus mytili De Morgan (Kidder, 1933). — The peripheral 
longitudinal fibrils linking the ciliary basal bodies originate from a 
transverse fibril in the anterior ventral region. These almost parallel rows 
of fibrils pass around the posterior end of the organism uninterrupted, 
continue over the anterior end, and again return to the transverse fibril 
of the ventral surface. Each basal body is furnished with a ciliary rootlet 
extending toward the endoplasm. 

An elongate mass of homogeneous material, the motorium, follows the 
posterior line of the cytopharynx and continues into the endoplasm as a 



CILIATE FIBRILLAR SYSTEMS 



231 




{lb. f. 



Figure 81. Chlamydodon sp. (MacDougall, 1928.) 

A. c. myon. — circular myoneme and traversing fibrils 

B. fib. f.- — fibrillar fan c. fib. — cross fibril 



b. gr. — basal granule 



Strand which appears to be made up of fibrils. This strand follows the 
posterior margin of the gullet and frays out in the endoplasm near the 
left side of the organism. From the outer end of the motor'ium originate 
two fibrils: one joins the basal elements of the posterior brush, while 
the other is the fiber of the dorsal peristomal row of cilia. The latter pro- 
ceeds anteriorly to join the anterior fibril and thus to the transverse fibril 
mentioned above. 



232 CILIATE FIBRILLAR SYSTEMS 

The fused basal elements of the large oral brush of cilia form a deeply 
staining band or plate at right angles to the long axis of the motorium. 
From its anterior end two fibrils are given off which join the anterior 
and middle oral brush plates. Fine fibrils arise from the basal elements 
of the large oral brush line of the cytopharynx and connect with the 
motorium along its anterior side. Finally, a ventral peristomal fiber runs 
under the basal plate of the long ventral peristomal cilia, curves slightly 
to the outside, and ends just anterior to the oral brush. 

The brushes of cilia about the mouth seem to connect directly with the 
motorium but not with the peristomal fbers. This lack of connection is noted 
in the movements of the cilia, the peripheral and peristomal cilia beating 
regularly, continuously and metachronously while the beating of the oral 
brushes is non-continuous and synchronous. 

Methods 

Fixatives: Flemming's, Zenker's (for whole mounts), Bouin's, Zenker's, 

strong Flemming's (for sections). 
Stains: Heidenhain's haematoxylin, Mallory's triple. 

Conchophthirus (Kidder, 1934). — The "well integrated and closely 
interconnected neuromotor systems" of three species of Conchophthirus 
— C. anodontae Stein, C. curtus Engl., and C. magna sp. nov. — are quite 
comparable. The description is given of the external, internal, and peri- 
stomal fibrillar complexes, with reference especially to C. magna. 

Most of the numerous, closely set ciliary rows originate in an antero- 
ventral suture and terminate in a dorsal suture near the posterior end. 
The ventral suture comprises two fibrils which are united at their ends 
and are connected irregularly by cross fibrils. This ventral suture is con- 
tinued posteriorly as the pre-orat connecting fiber, from which arise two 
fibrils: (1) connecting the rows of basal granules of the dorsal lip, and 
(2) connecting the basal granules of the pharyngeal ciliary row. 

The pre-oral connecting fiber itself becomes the peristomal net fiber. 
The latter gives off secondary fibrils to the peristomal field. On the left, 
these secondary fibrils are bounded by a longitudinal inner net fiber 
(Fig. 82) from which arise numerous fine fibrils that line the ventral 
side of the peristomal basket. On the floor of this basket, these fine fibrils 
join the inner basket fiber, which, in turn, gives off many branches that 
line the dorsal surface of the basket. These branches then unite with the 



CILIATE FIBRILLAR SYSTEMS 233 

fiber (noted above) that connects the basal granules of the dorsal lip. 
At the anterior, inner end of the pharynx is a large pharyngeal ring 
fiber that fuses with the inner basket fiber, previously noted, to form 
the fibrillar bundle. This point of fusion is regarded as comparable to the 
motorium of C. inytili. From the fibrillar bundle a gullet fiber extends 
inward and courses throughout the floor of the gullet, finally fraying out 
at the posterior end. 

The inner basket fiber is united posteriorly with the posterior basket 
connecting fiber. The latter, bending dorsally and to the right, comes to 
join the post-oral connecting fiber. Thus a direct connection is made 
between the peristomal region and the dorsal suture. From this post-oral 
fiber, numerous cross fibrils connect with adjacent ciliary rows. 

"This neuromotor system is thought to be mainly conductive but some 
parts of it may possibly be contractile or even supportive." 

Methods 

Fixatives: Klein's (1926), von Gelei-Horvath's (1931), strong Flem- 

ming's for four hours. 
Stains: Heidenhain's haematoxylin, destained with hydrogen peroxide. 



Dallasia frontata Stokes (Calkins and Bowling, 1929). — The most 
conspicuous part of the neuromotor system of Dallasia frontata was found 
to be the complicated apparatus of the mouth. This is composed of a 
tongue running through the buccal cavity, supported by bars which are 
anchored in long strands of dense material lying on the floor of the 
buccal cavity. There are two of these longitudinal strands and two series 
of bars from the tongue, one on each side. On the right and left sides 
are undulating membranes. A ladder-like organ originates anteriorly just 
below the membrane of the buccal cavity and at the right side of the 
mouth, and runs into the gullet. 

A discoidal mass on the left side of the gullet is interpreted as the 
motorium. It is connected by fibers directly to the proximal end of the 
tongue and strands. Similar fibrils connect the outer and the inner margins 
of the ladder-like structure with the motorium, and these fibers appear to 
form the outer and inner margins of this organ. Minute granules are 
present at the ends of each bar, at the points where the bars join with 
the longitudinal fibers. Posteriorly tivo fine fibers run from the motorium 



234 



CILIATE FIBRILLAR SYSTEMS 



gul. fib. 



ph. r. fib. 
fib. b. 



in. b. fib. 
d. f. p. b. 



-— i. 




p. o. c. 
ph. c. n 



i. n. fib. 
v.f.p.b. 



d. i.c.r. 

Z 

3 

p. b. c. f 
p. o. c. fl. 



Figure 82. Conchophthirus magna. (Kidder, 1934.) Diagram of the fibrillar system 
of peristomal region. 

d. f. p. b. — dorsal fibers of peristomal basket d. 1. c. r. — dorsal lip ciliary row 

fib. b. — fibrillar bundle gul. fib. — gullet fibril i. n. fib. — inner net fibril 

in. b. fib. — inner basket fibril 1, 2, 3, 4 — rows of body cilia 

p. b. c. f . — posterior basket connecting fibril p. o. c. f. — pre-oral connecting fibril 

p. o. c. f\ — post-oral connecting fibril ph. c. r. — pharyngeal ciliary row 

ph. r. fib. — pharyngeal ring fibril v. f. p. b. — ventral fibril of peristomal basket 

deep into the endoplasm, where they are lost, one in the vicinity of the 
macronucleus, the other near the contractile vacuole. 

Basal granules are described, but no connecting fibrils mentioned. 

The function of this enigmatical organ is purely conjectural; possibly it 
has something to do with the opening and closing of the mouth. . . . What- 
ever the specific function may be there is little reason to doubt that it is 
intimately connected with the irritability of the mouth region. 



CILIATE FIBRILLAR SYSTEMS 



235 



Method (whole mounts and sections) 

Fixatives: Schaudinn's, made up in 95 -percent ethyl alcohol, followed by 

prolonged treatment with turpentine. 
Stains: Iron haematoxylin ; solution of acid fuchsin and methyl green. 

Dileptus gigas (Visscher, 1927). — From a rod-like motorium found 
near the base of the gullet, several sets of fibrils extend to different 
parts of the body, as follows: (1) one set supplies the wall of the fun- 
nel-shaped gullet, (2) two more distinct fibrils course anteriorly along 
the proboscis, one on either side of a row of trichocysts; and (3) a set 
of branching finer fibrils spreads posteriorly over the body, where they 




mot. 



f, fib. 



Figure 83. Dileptus gigas. Neuromotor apparatus (camera drawing). (Visscher, 
1927.) 

a. iib. — anterior fibril f. fib. — fine fibrils i. fib. — inner fibrils 

mot. — motorium o. fib. — outer fibrils t. — trichocysts 



236 CILIATE FIBRILLAR SYSTEMS 

are probably associated with the longitudinal rows of cilia running paral- 
lel to the contractile fibrils (Fig. 83). 

Methods 

Fixative: Schaudinn's. 

Stains: Iron-haematoxylin, acid borax carmine. 

Entodiscus borealis (Powers, 1933). — The following distinct groups 
of fibrils and associated structures characterize the fibrillar system of 
Entodiscus borealis: (1) the stomatostyle, with its dorsal and ventral 
anterior horns and their adoral fibrils; the labial fibrils; the pharyngeal 
fibrils; and the circumpbaryngeal rods; (2) the anterior fibrillar center, 
or motorium, with its anterior, posterior, and marginal strands; the pos- 
terior auxiliary fibrillar center with its associated strands; and (3) the 
peripheral transverse commissural fibrils interconnecting the basal gran- 
ules. Closely associated with the commissural fibrils are the distal branch- 
ing ends of most of the internal fibrils. The ventral peripheral layer 
is further complicated by the long (6-12 p) ciliary rootlets, which ex- 
tend into the endoplasm from the basal granules of the cilia. 

The pellicle of the ventral surface is thicker than elsewhere and is 
highly difiPerentiated owing to pellicular fibrils accompanying each 
longitudinal ciliary row. These fibrils are conspicuous only at the anterior 
end. They are interpreted as either supporting or contractile in nature 
(Fig. 84). 

Methods 

Fixatives: Schaudinn's, Da Fano's, 2 5 -percent osmic acid, Flemming's 

without acetic acid (the two latter fixatives for sections). 
Stains: Iron alum haematoxylin, Yabroff's silver method. 

Entorhipidium echini (Lynch, 1929). — One fibrillar system of Etito- 
rhipidium echini consists of a motorium which is connected to a network 
of peripheral fibrils linking all the basal granules. The other set of fibrils 
is developed in the pellicle, chiefly on the ventral surface. 

The motorium is located to the left of the buccal cavity. It is com- 
posed of five heavy strands united into a rod-like body by longitudinal 
fibrils which appear to be continuous with the neurofibrils of the basal 
granules. These delicate longitudinal fibrils unite the basal granules of 
each peripheral row of cilia, and other fibrils encircle the body, uniting 



d. ad. fib. 
Q. fib. c. 
b.gr. 
i. m. fib. 



V. ad. fib. 
li . Gift, 
stom. 

ph. fib. 
r. m. fib. 




Figure 84. Entodiscus borealis. (Powers, 1933.) A. Reconstruction of neuromotor 
system, dorsal surface (modified from author). B and C. Relations of body cilia and their 
basal granules with fibrils of neuromotor system. 

a. fib. c. — anterior fibrillar center b. gr. — basal granule 

cil. r. — ciliary rootlet d. ad. fib. — dorsal adoral fibril 

1. m. fib. — left marginal fibril 1. pell. — longitudinal pellicular thickening 

li. cyt. — lips of cytostome p. fib. — posterior fibril 

p. fib. c. — posterior fibrillar center ph. fib. — pharyngeal fibril 

r. m. fib. — right marginal fibril stom. — stomatostyle 

t. f. — transverse fibril v. ad. fib. — ventral adoral fibril 



238 CILIATE FIBRILLAR SYSTEMS 

the basal granules with perfectly regular transverse commhsures. In the 
region posterior to the frontal lobe, the commissural fbrils branch, form- 
ing numerous collaterals extending in various directions. These unite 
with transverse and longitudinal fibrils. 

The pellicular thickenings of the frontal lobe are present in the form 
of heavy, deeply staining fibers which alternate with the ciliary rows. Be- 
tween the anterior ends of these and the anterior ends of the dorsal 
rows of cilia is a delicate fretwork, or polygonal area. The boundaries of 
polygons correspond with the boundaries of a double row of large 
vacuoles. Posterior to the frontal lobe, the heavy fibrils become fine and 
lie so close to the neurofibrils that they are almost indistinguishable from 
them. Similar fibrils are evident on the dorsal surface of the organism 
only at the anterior end (Fig. 85). 

Methods 

Fixatives: Schaudinn's, Da Fane's, 2-percent osmic acid, Flemming's with- 
out acetic acid (the two latter for sections) . 
Stains: Iron-haematoxylin, Yabroff's silver method. 

Eupoterion pernix (MacLennan and Connell, 1931). — Most of the 
longitudinal ciliary fibrils of the body take their origin from a heavy bar, 
the anterior connective fibril, that lies dorsoventrally across the anterior 
tip, whence they extend to the posterior end, where they fuse. Additional 
fibrils arise in pairs along the pre-oral suture line and these fuse mostly 
also at the posterior end. Each basal granule gives off one or two com- 
missural fibrils to adjacent ciliary rows, resulting in a fairly regular lat- 
ticework (Fig. 86). 

The neuromotorium lies beneath the wall of the cytostome. The fibrils 
of the four pairs of oral ciliary rows along the oral groove are fused in 
a V-shaped figure at the apex of the suture line. All of these end directly 
in the motorium or are closely connected to it by the transverse fibril or by 
the longitudinal ciliary fibrils. The transverse fibral lies across the end 
of the neuromotorium; its right end joins the pharyngeal fibrils, thus 
forming a pharyngeal strand; and its left end fuses with the two outer- 
most peristomal ciliary fibrils and ends farther left in a connective fibril 
of adjacent outer rows of cilia. The two rows of cilia arising from the 
anterior ends of the outer peristomial rows (of the ordinary peripheral 




1_ comm. 



Figure 85. A. Entorhipidium echini. (Lynch, 1929.) Cross section, showing periplast 
of the anterior dorsal fibril. B. Entorphidium echini, tangential section of anterior ventral 
surface. 

A. b. gr. — basal granule B. comm. — commissural neuro-fibril 

1. p. fib. — longitudinal pellicular fibril I. fib. — longitudinal fibril 

t. — trichocyst p. fib. — pellicular fibril 

tr. fib. — transverse fibril 



240 CILIATE FIBRILLAR SYSTEMS 

type) are connected to the rest of the body rows by commhsural fibrils, 
thus uniting the peristomiai ciha with the rest of the body ciHa. 

Methods 

Fixative: Schaudinn's (60° C). 
Stain: Heidenhain's iron haematoxylin. 

Haptophrya michiganensis Woodhead (Bush 1934). — Haptophrya 
michiganensis has an integrated system of fibrils that center in a moto- 



ph. sir. WC, 



I if. fib. 
^-z---ph.fib. 
/?. mot. 



Figure 86. Eupoterion pernix, optical section. (MacLennan and Connell, 1931.) 

a. c. fib. — anterior connective fibril n. mot. — neuromotorium 

b. gr. — basal granule ph. fib. — pharyngeal fibril 
cyto. — cytostome ph. str. — pharyngeal strand 
ectopl. — ectoplasm tr. fib. — transverse fibril 

rium. Within the sucker at the anterior end is a fibrillar ring, homologous 
with the esophageal ring of stomatous ciHates. The uiolovium is located 
in the center of this ring and is connected with it by radial connectives. 
Accessory bodies of the motoriuni are suspended from various points on 
the inner edge of the ring. (These are not evident during fission.) Nu- 
merous myonemes radiate from the inner edge of the fibrillar ring and 
extend to the opposite walls, posteriorly and laterally, dividing into 
fine fibrils at their outer ends. Equally spaced peripheral myonemes arise 
from the external edge of the fibrillar ring, adhere to the inner layer of 



CILIATE FIBRILLAR SYSTEMS 241 

the ectoplasm, and extend to the posterior end of the animal. These 
myonemes are "closely associated with the basal granules." Commissures 
connecting the basal granules form a close network over the entire body. 
Supporting fibrils from the nuclear membrane, the endoplasmic cone, and 
the contractile canal extend to the peripheral ectoplasm (Fig. 87) . 

The deeply staining mass was interpreted as a niotorium because "" (1 ) 
it is connected, directly or indirectly, with all parts of the fibrillar system; 
(2) it is near the anterior end of the ciliate; (3) if, with this mass, the 
sucker is removed, the animal loses its power of worm-like forward 
movement even though the cilia continue to beat; and (4) a toxic sub- 
stance acts first upon the anterior part, particularly the sucker, where- 
upon the animal ceases its forward movement." 

Methods 

Fixatives: Schaudinn's and Zenker's. 

Stains: Delafield's and Heidenhain's haematoxylin ; also Kolatschev's osmic 
impregnation, as outlined by Bowen ; Yabrofif method. 

Ichthyophthirius (MacLennan, 1935). — The longitudinal fibrils con- 
necting the basal granules beneath the ciliary rows of the body surface 
are linked together anteriorly and to some extent posteriorly by small 
centers, the anterior and the posterior fields. The centers are connected 
by a "suture fibril" which marks the ventral side. The suture fibril is 
interrupted by the oral region, thus dividing it into pre- and post-oral 
segments. Concentric accessory suture fibrils lie on the sides of the oral 
region and terminate anteriorly and posteriorly in the suture fbrils. The 
lip of the oral opening is bounded by ( 1 ) the outer peristomal fibrils, 
which are also linked to the suture fibril. (2) Circular fibrils line the 
walls of the oral cavity and radial fibrils intersect the two sets of fibrils 
at right angles. These transverse connections between the ciliary fibrils 
are present only in the oral region. 

Two heavy basophilic rods, each attached to a heavy esophageal fibril 
are located near the esophageal plug. An inner peristomal fibril runs from 
this bilobed neuromotorium to the basal granules. 

Ciliary rootlets are developed in the region of the inner peristomal 
fibrils; they are less well developed in the region of the outer peristomal 
fibrils, and not found in the region of ordinary body cilia. About 50-100 
individual ciliary rootlets combine to form numerous esophageal strands. 




con t. con, fib. 
cont. can. 



nuc. m. 
nuc. fib. 

- macro. 
. _ end pi. fib. 
-^endopl. c. 



rod. my. _ 



per. m. __ 



b.gr. 




_._ c. fib. 
re-t. fib. 

rod. conn. 

mob. 

...flbnn 

-Qcc. mot. 



comm. -4 v 



Figure 87. Haptophrya michiganensis. (Bush, 1934.) A. Diagrammatic section show- 
ing one-fourth of the animal body with part of the macronudeus. B. Diagram of anterior 
part of neuromotor system, 

ace. mot. — accessory motorium mot. — motorium 

b. gr. — basal granule macro. — macronudeus 
comm. — commissures nuc. fib.— nuclear fibrils 

c. fib. — coarse fibrils nuc. m. — nuclear membrane 
cont. can. — contractile canal pell. — pellicle 

cont. can. fib. — contractile canal fibrils per. my.^ — peripheral myoneme 

endopl. c. — endoplasmic cone rad. my. — radial myonemes 

endopl. fib.- — endoplasmic fibrils rad. conn. — radial connectives 

fibr. r. — fibrillar ring ret. fib. — reticulate fibrils 



CILIATE FIBRILLAR SYSTEMS 243 

each of which turns sharply and continues to the endoplasm, parallel to 
the main axis of the oral pit. 

Method 

Fixatives: Schaudinn's, Zenker's. 

Stains: Heidenhain's iron-haematoxylin, Delafield's (for fibrils), Klein's 
(1926) (for ciliospores), Lund's wet silver method (for adults). 

Lechriopyla mastax (Lynch, 1930) . — The cilia of Lechriopyla mastax 
can be divided into three areas : ( 1 ) the cilia of the general surface of 
the body, (2) the cilia of the peristome, and (3) a transverse band 
of cilia known as the supraoral band. The basal granules of all these 
cilia are connected by delicate longitudinal fibrils without transverse con- 
nectives. Below the ciliary lines of the peristome and vestibule are verti- 
cal pellicular lamellae, which are fused to the furcula described below. 

A crescent-shaped neuromotorium lies beneath the pellicle at the 
left end of the peristome. Its ends are continued as long fibers, the 
anterior and posterior adoral fibers, which form a complete (or nearly 
complete) ring about the peristome. From the ring arise the ciliary lines 
which extend over the surface of the body, or pass into the peristome 
and the pharyngeal involution. A variable number of fibrils arising from 
the outer border of the motorimn extend through the cytoplasm for vary- 
ing distances, to fuse with the pellicle just beneath a ciliary line. They 
may branch or anastomose, and they become more delicate distally. 

The jurcula, shaped much like a heavy tuning fork, partly surrounds 
the vestibule. The ends fuse with the walls of the pharynx, and delicate 
fibrils from the pharyngeal wall extend to the jurcula. This organelle 
may be an additional element of the neuromotor system. 

A long pellicular fiber extends from the left end of the internal open- 
ing of the cytopharynx to the middle of the posterior end of the organism 
and occasionally curves along the right side for varying distances. This 
fibril is not included in the author's description of the neuromotor sys- 
tem. 

Methods 

Fixatives: Bouin's, Schaudinn's, Da Fano's cobalt nitrate-formalin. 
Stains: Iron alum haematoxylin, carmines, cochineals, Yabroff's silver- 
nitrate method (no success), Klein's silver method. 
Sections prepared in a variety of ways. 



244 CILIATE FIBRILLAR SYSTEMS 

Ptychostonium chattoni Rossolimo (Studitsky, 1932).- — The mouth of 
this parasitic ciiiate is at its posterior end. At the anterior end is a horse- 
shoe-shaped sucker with a projecting rim for attachment. 

The sucker [Fixationsapparat) is provided with a system of fibrils. 
The largest fibril, the peripheral cord, borders the sucker and gives off 
at its ends fine fibrils that extend into the cytoplasm. The sucker's disc 
has four sets of fibrils: ( 1 ) the deeper set comprises two groups of paral- 
lel fibrils that cross each other at a sharp angle as they traverse the disc, 
both groups coming to adhere to the peripheral cord; (2) the uppermost 
set, visible in the living organism, courses from right to left and from 
anterior to posterior; (3) a third set of fine fibrils are attached to the 
peripheral cord by their anterior ends; and (4) the fourth set, com- 
posed of sixteen or seventeen strands that run from right to left and 
from anterior to posterior, frays into four or five fine fibrils at the anterior 
end of each strand, to become attached to the peripheral cord and to 
other adjacent fibrils; the posterior ends of these strands become fimbri- 
ated also into fine fibrils. All apparently serve for support. 

Methods 

Fixatives: Schaudinn's, Carney's, Bouin's, Champy's and Benda's (for 

whole mounts), Altmann-Kull's (for sections). 
Stains: Heidenhain's haematoxylin, safranin, von Gelei's toluidin blue, 

Mallory's triple. 

B. HETEROTRICHA 

Balantidium coli Malmsten and B. suis sp. nov. (McDonald, 1922) . — 
The motortum of Balantidium suis lies within the ectoplasm of the 
apical cone, close to the right ventral wall of the esophagus. A fibril 
encircling the esophagus arises and ends at the anterior end of the moto- 
rium, where the adoral ciliary fiber also arises. The circuniesophageal 
fibril has irregular enlargements, from which fibers pass both posteriorly 
and anteriorly into the ectoplasmic mass of the anterior end. These fibrils 
appear to fade out in the ectoplasm. The adoral fiber connects the basal 
granules of the adoral cilia. The remainder of the fibrils are not directly 
connected with the motorium. 

The basal granules of the peripheral longitudinal spiral rows of cilia 
are so closely set that it has been impossible to see a fibrillar connection. 
No transverse fibrils connecting the rows were observed. A ciliary rootlet 



CILIATE FIBRILLAR SYSTEMS 245 

extends from each basal granule, and a second small granule is found 
at the junction of the ectoplasmic and endoplasmic layers. The ectoplasmic 
layer is quite thin, except at the anterior end of the organism, where it 
is deep and the distance between the granules of each cilium correspond- 
ingly long. The rootlets of the row of adoral cilia around the margin of 
the peristome, the "radial fibrils," are exceedingly long, ending in about 
the posterior third of the body without connection or attachment. The 
cilia immediately posterior also have long rootlets, but they become 
shorter as they approach the base of the apical cone (Fig. 88) . 

Methods 

Vital stain: Neutral red (differentiates the neuromotor apparatus). 
Fixatives: Schaudinn's, Zenker's, Formalin, osmic acid, picrocuric, 

60-80° C. 
Whole mounts and sections 
Stains: Iron haematoxylin, Mallory's triple stain (particularly for sections). 



pe//. ___ 
b.gr. --- 
cil.r. 




/ 



Figure 88. BaLvitidium coli, cross section of peripheral region. (McDonald, 1922.) 
b. gr. — basal granule gr. b. — granular band 

cil. r. — ciliary rootlet hy. b. — hyaline band 

endo. — endoplasm pell. — pellicle 

Balantidium sushilii (Ray, 1932). — Fibrils associated with the con- 
tractile vacuoles have been described in Balantidium sushilii. Each of the 
two lateral vacuoles has a fibril running from the wall of its outer half 
to the neighboring pellicle. The neck of the terminal vacuole is sur- 
rounded by a diaphragm of fibrils running from the wall of the neck 
to the surrounding pellicle. These unusual fibrils are described and figured 
in the extended as well as the contracted condition. 

An axial and a peripheral system of fibrils can be seen also in the 
living organism. Stained preparations show that the former consists of 



246 CILIATE FIBRILLAR SYSTEMS 

three or four fibrils, parallel or twisted after the manner of a rope, which 
originate below the pellicle at the anterior end and extend posteriorly 
a short distance beyond the mouth. The individual fibrils gradually be- 
come thinner toward the posterior end, where they come in contact with 
the limiting membrane formed by some of the fibrils of the peripheral 
system. At the anterior termination of these fibrils is found a knob-like 
structure which may be imbedded in the epithelium of the host. 

The peripheral system of fibrils is arranged in two conspicuous arches 
along the left anterior border of the organism. Anteriorly, fibrils extend 
both to the right and to the left peristomal lips. Posteriorly, the two 
arches converge. Some of the fibers continue mesially and come to form 
a kind of limiting membrane beyond which no fibrils are traceable. 

The group of fibers forming the axial system, together with the borer 
attached at its anterior end, is called the boring apparatus, as the first 
of its kind to be noted. It may be compared with the axostyle of some 
flagellates. 

The peripheral system is believed to represent morphonemes. 

Methods 

Fixatives: Brasil's modification of Bouin-Duboscq's (for whole mounts), 

Bouin's alcoholic, twenty-four hours (for sections 5 \i.) . 
Stai)i: Heidenhain's haematoxylin (whole mounts and sections). 

Fabrea salina Henneguy (Ellis, 1937). — In Fabrea there are numer- 
ous longitudinal rows of closely set body cilia (in pairs). These are 
interrupted on the ventral side by a coiling adoral zone. The basal gran- 
ules are connected by fine longitudinal fibrils. No transverse connections 
or ciliary rootlets were observed. Each membranelle consists of two rows 
of basal granules whose ciliary rootlets fuse into a single plate, the basal 
lamella. Each longitudinal fibril of the dorsal and ventral surfaces, with 
the exception of those that merge with one another, is connected at the 
adoral zone with the basal lamella of a membranelle. The basal lamella 
is connected with the adoral fibril by fibril running across the peristomal 
groove. The adoral fibril starts at the anterior tip and follows the course 
of the inner border of the adoral zone. The peristomal fibril is continued 
beyond the end of the adoral zone on the wall of the funnel and ends 
in a ganglion-like body on the left wall of the ventral lobe. From this 
motorium arise several fibrils — the adoral fibril, which follows the course 



CILIATE FIBRILLAR SYSTEMS 247 

of the adoral zone, and other fibrils which appear to end bhndly in the 
endoplasm of the ventral lobe. Anteriorly the fibrils of the frontal field 
tend to converge and end very obliquely on the adoral fbril. 

No pellicular pattern was demonstrated by any of the silver methods, 
but they did show the longitudinal fibrils connecting the cilia. 

Methods 

Fixatives: Schaudinn's, Bouin's and Flemming's. 

Stains: Iron-haematoxylin, Mallory's triple. 

Silver method : Yabrotf's modiiication of Da Fane's. 

Metopus circumlahens (Lucas, 1934). — The motorium of Met opus 
circiiuilabens lies posterior to the cytostome. From its left side it gives rise 
to a pair of ventral adoral fibrils which follow the peristomal curvature 
outward to the oral margin and there end in a sort of arborization. Each 
row of peristomal membranelles arises immediately in contact with a con- 
nective between these two fibrils. A dorsal adoral fibril extends from the 
right side of the motorium. At slightly irregular intervals is gives rise 
to from ten to twenty heavy connectives, which may partially fuse in 
pairs as they curve beneath the dorsal wall of the peristome to its left 
side. There they turn ventrad and unite with the ventral adoral fibers. 
A fibrillar pharyngeal strand arises from the posterior end of the moto- 
rium. Its course varies in different organisms, but it usually lies along the 
right lateral wall of the organism to the right of the cytopharynx. Near 
the posterior end of the latter structure it forms a large spiral coil. 

The entire body surface, except the right lateral margin, is covered 
with rows of cilia. Longitudinal ciliary fibrils are present, but no com- 
missural fibrils were observed. Each basal body in the most dorsal of the 
five rows of the crest cilia gives rise to rootlets which end freely in the 
cytoplasm. A relation between these peripheral fibrils and the fibrils of 
the motorium seems to be suggested by the numerous fine branches which 
arise from the ventral adoral fibrils. These extend indefinitely into the 
cytoplasm toward the longitudinal ciliary rows of the ventral surface. 

In view of the contrastingly striking and obvious specialization in the 
fibrillar structure of the neuromotor system about the peristomal, pharyngeal, 
and central endoplasmic regions of the cell, one is inclined to believe that 
the neuromotor system of this ciliate is vitally, though not exclusively, con- 
cerned in the conductile functions related to the metabolic activities of the 
organism. It is possible that, because they are located within the mobile 



248 CILIATE FIBRILLAR SYSTEMS 

cytoplasm of the protozoan, the stouter of these various fibres may serve in 
addition some function in the nature of support. 

Methods 

Fixatives: Schaudinn's, Bouin's, Jorgensen's, Van Rath's. 
Stains: Heidenhain's iron haematoxylin, Regaud's haematoxyhn, Mallory's 
triple (whole mounts and sections). 

Nyctotherus hylae (Rosenberg, 1937). — A detailed description of the 
neuromotor system of Nyctotherus hylae is given, including two centers 
and a group of special fibrils believed to control the reversal of ciliary 
action. An incomplete account of some of these structures was given by 
Kirby (1932) for N. sylvestrianus. The movements of N. hylae were 
studied by cinematographic methods. 

From the main motorium located at the distal end of the cytopharynx 
arise two sets of unbranching fibrils. One set extends along the anterior 
border of the pharynx, eventually terminating at the end of the peristome. 
The other set follows the posterior border and arm of the ectoplasmic 
thickening. Some of the latter fibrils unite at the cytostomal border with 
the peripheral peristomal fibril. The so-called "reversal fibrils" originate 
from the posterior part of the motorium, radiate through the endoplasm, 
and at their distal ends unite with the ciliary lines at several points, not 
including the presutural ciliary lines. 

The membranelles have each a basal plate and two rows of basal gran- 
ules, from which fine fibrils connect with the circumpharyngeal fibrils. 
The latter become the transverse peristomal fibrils, of which there are at 
least two for each membranelle of the series. 

A number of fibrils from the motorium directly connect with the 
anterior neuromotor center. From this structure arise many ciliary lines 
that connect rows of basal granules. Commissural fbrils between these 
lines are present only in the apical post-sutural region. The lateral and 
sagittal sutures which divide the ciliation into definite regions were 
interpreted as probable conductors between ciliary lines. 

The pharyngeal ter^ninus, a. deeply staining structure, gives rise to a 
post- pharyngeal bundle of fibrils that have no apparent distal attach- 
ment. Kirby (1932) described a similar "band formed structure" in 
N. silvestrianus which may extend beyond the cytopharynx, its course 
in the endoplasm varying in dififerent individuals. He considers it "ho- 



CILIATE FIBRILLAR SYSTEMS 



249 



mologous with the 'continuation tube' of the "subpharyngeal canal' de- 
scribed by Higgins (1929) in N. cordiformis" (Kirby, 1932, p. 298). 
Whether or not this strand is a part of the neuromotor system is an 
open question. 

Fibrils interpreted as nior phonemes are as follows: (1) those extend- 



comm 



. fib. 



tr. perist. fib. 



a. n. c. 
c.b.b. 



lai.sui. 
n. env. 



per. pehst fib. 

cir. fib. 
p. /. ph. fib. 

mob. 
sk f fib. 




-ph. term. 
_ re. fib. 



Figure 89. Nyctotherus hylae, fibrillar system, diagrammed. (Rosenberg, 1937.) 



ant. neur. cen. — anterior neuromotor cen- 
ter 
c. b. b. — ciliary basal body 
c. 1. — ciliary line 
ca. fib. — caryophore fibril 
cir. fib. — circumpharyngeal fibril 
comm. fib. — commissural fibril 
1. ph. fib. — longitudinal pharyngeal fibril 
lat. sut. — lateral suture 
mot. — motorium 



n. env. — nuclear envelope 

p. 1. ph. fib. — posterior longitudinal 
pharyngeal fibril 

p. ph. b. — post-pharyngeal bundle 

ph. term. — pharyngeal terminal 

per. perist. fib. — peripheral peristomal fi- 
bril 

re. fib. — reversal fibrils 

sh. s. fib. — shelf supporting fibrils 

tr. perist. fib. — transverse peristomal fibril 



250 CILIATE FIBRILLAR SYSTEMS 

ing from the right to the left of the body, (2) the caryophore fibrils, 
and (3) the shelf-supporting fibrils (Fig. 89). 

Methods 

Fixatives: Schaudinn's (5-percent acetic), Fiemming's. 

Stain: Heidenhain's iron haematoxylin, aqueous and alcoholic. 

Silver techniques: Klein, Gelei-Horvath, Yabroff (negative). 

Spirostonium anibiguum Ehrbg. (Bishop, 1927). — The ridges and 
furrows in the ectoplasm of Spirostonium ambiguum follow a sinistral 
spiral course from the anterior to the posterior end of the body. Be- 
neath the furrows lie thread-like myonevies, somewhat beaded in ap- 
pearance, but without light and dark alternating bands. The rnyonenies 
taper gradually as they approach either end of the body and finally disap- 
pear from view. They are not attached to any structure. Longitudinal 
rnyonenies were found on either side of and running parallel to the band 
of peristomal membranelles, except along those membranelles nearest 
the cytostome. No evidence was obtained confirming the presence of 
other fibrils such as neurophanes. 

On the anterior side of each niyoneme lie the basal granules of the 
body cilia. The rows of granules are parallel to and slightly above the 
level of the rnyonenies. No ciliary rootlets nor connections between basal 
granules or myonemes were discovered. 

The system of fibrils underlying the membranelles (Fig. 90) of 5. 
ambiguu?n includes: 

an anterior basal fibril extending from the anterior end of the body to the 
beginning of the peristomial depression ; a middle fibrillar system which 
varies in its course in diiferent individuals, but which collects the end-threads 
of the membranelles lying on the left side of the peristomial depression ; and 
a posterior basal fibril into which the end-threads of the membranelles at 
the posterior end of the peristomial depression and in the cytopharynx join. 
A connection between the posterior basal fibril and the middle fibrillar 
system is seldom found, and there is always a break between the middle 
fibrillar system and the anterior basal fibril. 

A central body to which the fibrils join was found in no case. 

Methods 

Fixatives: Schaudinn's, picro-mercuric (hot). 

Stains: Iron-haematoxylin (alcoholic and aqueous), Mallory's triple 
(Sharp's modification), Fuchsin S. 



CILIATE FIBRILLAR SYSTEMS 



251 




memb. 



KA_,,b.pf. 



b.i. 



Figure 90. Spirostoiiium 
ambiguum. Diagram of 
membranelles and their 
intracytoplasmic structures. 
(Bishop, 1927.) 
b. fib. — basal fibril 
b. 1. — basal lamella 
b. pi. — basal plate 
e. thr. — end thread 
memb. — membranelle 



-_e. thr. 
^ b. fib. 

C. OLIGOTRICHA 

Diplodinlum ecaudatum (Sharp, 1914). — The motorium is located 
in the ectoplasm above the base of the left skeletal area and between the 
left extremities of the dorsal and the adoral membranelle zones. Dorsally 
it is connected with the bases of the dorsal membranelles by a dorsal 
motor strand, which also sends a branch along the base of the inner 
dorsal lip. A ventral motor strand connects the motorium with the bases 
of the adoral membranelles and its branch and passes along the base 
of the inner adoral lip. Numerous fibers from the motorium follow the 
contour of the operculum and disappear near the base of the right skeletal 
structure. A circumesophageal ring surrounds the esophagus at the level 
of the outer adoral furrow, from which a fibril connects with the mo- 
torium. Certain fibrils in the wall of the esophagus appear to unite with 
the circumesophageal ring; others are attached to skeletal structures and 
are considered contractile fibrils. Rootlets from the oral cilia end in, or 
close to, the ring. A coordinating (conductive) function was ascribed 
to this fibrillar system of D. ecaudatum, because of its initimate relation- 
ships with the motor organelles and its complete structural integration 
through the motorium. 

Methods 

Fixatives: Schaudinn's (alcoholic, hot), Zenker's, Flemming's, Worcester's, 
Bouin's, formalin 4 percent, osmic acid one percent (formalin 36° C.) 
Stains: Heidenhain's haematoxylin and Mallory's triple. 



252 CILIATE FIBRILLAR SYSTEMS 

Diplodinium ruedimn (Rees, 1931). — The structure of D. medium is 
compared with D. ecaudatum as described by Sharp. The disagreement 
between Rees's interpretation and the interpretation of Sharp has to do 
primarily with the ectoplasmic layer directly under the pellicle, except 
in the region of the adoral lip and the inner boundary layer of the ecto- 
plasm. In D. medium this layer, according to Rees, is more prominent 
than in D, ecaudatum and consists, instead of fine alveoli, of an inter- 
woven network, or complex system of fibrils. Serial cross and longitudi- 
nal sections, 3 pi. in thickness, made it possible to trace the ectoplasmic 
layers. 

Cross sections of D. medium show a fold of this middle layer of the 
ectoplasm which corresponds in its position to Sharp's motorium. Further- 
more, the esophageal ring described by Sharp is interpreted as section 
of the inner boundary layer of ectoplasm. The fibers connecting the 
mortorium with the membranelles and esophagus could not be differ- 
entiated. 

The ciliary rootlets are attached to membranes composed of sheets of 
the fused middle and inner layers of ectoplasm, which in turn are at- 
tached to the fibrillar system of the ectoplasmic layers The structures are 
membranes, according to Rees, because "one or the other of them occurs 
in all longitudinal and oblique sections, whether cut with reference to 
the parasagittal plane or to a plane at right angle or at any other angle 
to it." The esophageal tractor strands are considered to be a part of the 
ectoplasmic network of fibrils. The membranes, instead of strands, are 
believed by Rees to function in the retraction of the adoral and the dorsal 
cilia. 

The occurrence of fine fibrillae in the non-ciliated ectoplasm of Diplodinium 
is of interest in connection with other papers on the neuromotor system. It is 
obvious that in the latter ciliate the fibrillae of the ectoplasmic layers have no 
relationship to a neuromotor system. 

Methods 

Fixatives: Not listed. 

Stains: Iron-haematoxylin, Zirkle's N-butyl alcohol method of dehydration. 

Diplodinium Schbg. (Kofoid and MacLennan, 1932). — In addition 
to the neuromotor apparatus as described by Sharp (1914) for D. ecauda- 
tum and incidentally confirmed in this systematic investigation, a fibrillar 



CILIATE FIBRILLAR SYSTEMS 253 

complex also was found to occur in the caudal spines of D. dentatum, 
in contrast to the apparently structureless spines in Entodinium. Along 
the bases of the caudal spines appeared a heavy marginal fibril from 
which finer anchoring fibrils extended into each spine, terminating under 
the cuticle of its outer margin. A very heavy main anchoring fibril 
bordered the inner edge of each spine. From the anchoring fibril of the 
ventral spine smaller fibrils branched toward the anus, where they ended, 
one on each side. Small branches from these coursed in the wall of the 
rectum, parallel to its main axis. 

No connection was evident between this fibrillar complex of the caudal 
spines of D. dentattwi and its neuromotor system. The location and re- 
lationships of the former suggested a supporting function similar to that 
of the longitudinal surface fibrils. Also, since these spines undergo a 
change in their curvature such as might obviously be facilitated especially 
by the main anchoring fibril together with the marginal fibrils, these 
caudal fibrils were considered to be myonemes. 

A similar fibrillar system had been described by Belaf (1925) in 
Epidinium caudatum, on the basis of which Reichenow (1929) denied a 
neuromotor function for all fibrils of the Ophryoscolecidae. The clear 
difference in the morphological relationships of the two fibrillar systems 
in D. dentatum, however, indicated that these systems have quite difi^er- 
ent functions: 

The caudal fibrils are admirably situated to serve as supporting and contractile 
structures. The motor fibrils are so situated as to be of little or no use 
either as supporting or as contractile fibrils. The caudal fibrils show no con- 
nection to the motor organelles. The motor fibrils link together (through the 
neuromotorium) all the motor organelles of the individual. 

Methods 

Fixative: Schaudinn's. 
Stain: Iron-haematoxylin. 

Favella jorgensen (Campbell, 1927). — The netiromotorium is a 
spindle-shaped body in the ventral ectoplasmic wall in the mid-region 
of the gullet. This organelle gives rise to five intracytoplasmic fibrils as 
follows: ( 1 ) the adoral fibril, extending to and interconnecting the mem- 
branelles; (2) the circmnesophageal fibril, with branches surrounding 
the gullet; ( 3) a dorsal fibril which appears to connect with the striations 



254 CILIATE FIBRILLAR SYSTEMS 

of the oral plug; and (4, 5) two ventral fbr/ls extending downward 
and ending freely in the endoplasm. It was observed that the mem- 
branelles not only serve the organism in feeding and in locomotion, but, 
during periods of binary fission, function in the building of the lorica. 
In addition to their fibrillar connection with the motorium, each mem- 
branelle is supplied with three large basal bodies. 

Method 

Fixative: Schaudinn's (aqueous and alcoholic), 90° C. 
Stain: Iron-haematoxylin (whole mounts and sections). 

Tintinnopsis nucida (Campbell, 1926). — The somatic ciliation is con- 
fined to the column and forms in longitudinal rows along the myonemes. 
These showed basal granules, but without fibril connection. The myo- 
nemes are ectoplasmic structures, arranged longitudinally and un- 
branched. Anteriorly, they extend to the reflexed margin of the collar 
and possibly connect with basal granules of the adoral membranelles; 
posteriorly, they fade out. 

At the base of each adoral membranelle are three basal granules con- 
nected by fibrils. Through this triangular base passes the adoral motor 
fbril. Three oral membranelles (flat plates of fine cilia) follow the 
spiral of the gullet. Each oral membranelle ends in a distinct basal body. 
The ciliary membrane (undulating membrane of unusual construction), 
which functions in house-building and repair, is connected through its 
basal granules to the adoral fiber. A retractile tentaculoid is found be- 
tween each adoral membranelle. Tentaculoids, accessory combs, and 
trichocysts have no known fibrillar connection. 

The motorium is located in the ectoplasm of the ventral wall of the 
column. From it arise directly (1) the adoral fiber (granular), which 
connects with the adoral membranelles, the oral membranelles, and the 
ciliary membrane; (2) two dorsal fibers, extending into the ectoplasm 
adorally, where they end freely; and (3) the ventral fiber, which extends 
downward and also ends freely in ectoplasm. The circumesophageal ring 
is connected to the motorium indirectly by a single fiber. Short fibers 
from the ring surround the gullet. 

Methods 

Fixatives: Schaudinn's, 90° C. 

Stain: Heidenhain's iron-alum haematoxylin, aqueous and alcoholic. 



CILIATE FIBRILLAR SYSTEMS 



255 



D. HYPOTRICHA 

Oxytricha (Lund, 1935). — The parts of the neuromotor system of 
Oxytrkha apparently are confined to the more speciahzed organelles. No 
fibrils were found in connection with most of the ventral cirri. 

A long membranelle fibril connects the inner ends of the membranelle 
plate. The frontal membranelles, in addition, have ciliary rootlets which 
arise only from the most proximal basal granules of each of the three 
rows. These combine into a stouter fibril for each membranelle, which 
ends free in the endoplasm. 

Along the ventral margin of the peristome are numerous fibrils (Fig. 
91). One of these connects the basal granules of the undulating mem- 

memb.r. - 

b.pl.memb..- 
term. fit. 




ect.f. 



Figure 91. Oxytriclja. Diagram of fibrillar complex of cytostome, ventral view. 
(Lund, 1935.) 

b. fib. u. memb. — basal fibril of undulating membrane 

b. pi. memb. — basal plate of membranelle d. cy. fib. — dorsal cytostomal fibril 

ect. f.: — ectoplasmic fold memb. r. — membranelle rootlet 

mg. fib. memb. — marginal fibril of membranelles 
mg. u. memb. — position of marginal undulating membrane 

p. e. fib. — post-esophageal fibril term. fil. — terminal filament 

V. cy. fib. — ventral cytostomal fibril 



256 



CILIATE FIBRILLAR SYSTEMS 



brane. Twenty-two originate near the anterior end of this fibril. Their 
posterior destinations are as follows: ten terminate in ten small granules 
attached to the marginal fibril in the region of the posterior seven or 
eight membranelles; six pass along the dorsal wall of the gullet and 
extend into the endoplasm to a point near the right side of the body; 
and the other six continue into the gullet to form the ventral post-esopha- 
geal fibr/ls. These fibrils along the right side of the peristome were ob- 
served to be lax, apparently nonelastic, and capable of individual move- 
ment. 

A delicate fibril extends anteriorly from each of the five anal cirri. 
In the region at the left of the posterior macronucleus they disappear 
from view. Their position suggests, however, that they may join the 
other parts of the neuromotor system, in the region of the posterior im- 
dulating membrane fibril. 

Methods 

Fixatives: Schaudinn's (with 5-percent acetic), Zenker's. 




x...mot. 



Figure 92. Uroleptus halseyi, section through anterior end. (Calkins, 1930.) 
b. gr. — basal granule c. fib. — coordinating fibrils 

b. pi. — basal plate mot. — motorium 



CILIATE FIBRILLAR SYSTEMS 257 

Stains: Iron-haematoxylin, Mallory's triple (Sharp's modification). 

AUcrodis'section 

U role plus halseyi Calkins (Calkins, 1930). — The conspicuous parts 
of this kinetic system are the motoviinn near the right side of the gullet 
and, leading from it, the longitudinal anterior fibril which links a row of 
endoplasmic granules. Each basal plate of the membranelle series is con- 
nected by a short fibril to one of these basal granules, in regular order, 
and additional connectives unite the basal granules of each frontal cirrus 
with the chain. One short anterior fibril from the motorium extends to 
the margin of the peristome; the other leads to the undulating membrane, 
and two posterior fbrils are soon lost in the endoplasm. (Fig. 92) . 

Methods 

Fixatiies: HgCl,, saturated in 95-percent alcohol. 
Stain: Iron-haematoxylin. 

Conclusions 

It is evident from the review presented in foregoing paragraphs that 
the differentiation of fibrils in ciliates has been established for various 
representatives of their major groups beyond any doubt. Such differ- 
entiations, as revealed in fixed and stained material, are not artifacts, 
for many may be seen in living or in slowly disintegrating organisms 
(Worley, 1933). 

It is not certain, however, that all of the structures thus identified are 
actually fibrillar. Some may represent rather a sculptural, fibrillar-like 
pattern in the pellicle. 

It is also clear that the fibrils are not all alike, either structurally or as 
related to other protoplasmic differentiations of the cell. This was well 
illustrated in the several complexes of fibrils in the contractile stalk 
of the vorticellids. Here the structural elements composing the Spironeni, 
for example, differed partly in kind, but especially in arrangement, 
from those of the Axonem. Also, their relations to the fibrillar com- 
plexes of the bell were found to be different. 

Again, it is known that many fibrillar complexes of various ciliates 
are intimately associated with the basal apparatus of the motor organ- 
elles. This was shown for the inner fibrillar complex, or the injracilia- 



258 CILIATE FIBRILLAR SYSTEMS 

ture, of holotrichs such as Paraniec'nnn; for the membranelle fibril of 
Stentor and of Euplotes; and for most of the fibrillar systems of the other 
ciliates, the accounts of which were more briefly reviewed. 

Also, the literature contains numerous records of fibrils, in a variety 
of ciliates, which are structurally integrated into a so-called fibrillar 
system. It is with such fibrillar systems that this review has been chiefly 
concerned. 

Having established the identity of these fibrils and fibrillar systems 
and, for many, their structural continuity or contiguity with other organ- 
elles of the cells, especially the motor organelles, the investigators' further 
interest has, of course, been concerned with the function or functions 
which may be performed by such definitely related and integrated fibrillar 
systems. 

It was previously pointed out that the interpretation of the function 
or functions of these fibrillar systems has been based largely on the evi- 
dences of their structural integration and their relationship to other 
organelles. Relatively few of the interpretations have been made from 
experimental evidence. From both kinds of evidence, it was noted that 
at last four elementary functions have been ascribed by the many in- 
vestigators to these various fibrils or fibrillar systems : ( 1 ) elasticity, ( 2 ) 
mechanical support, (3) contractility, and (4) conductivity. 

Some examples of these included ( 1 ) Elasticity, the Spasnioyiem and 
pellicle in the contractile stalk of the vorticellids (Entz, 1893), the axial 
filament of cilia (Koltzoff, 1912); (2) Mechanical support. Stiitzgitter 
system of Paramecium (von Gelei, 1929); fibrils generally (Jacobsen, 
1931); (3) Contractility, myonemes of Stentor (Johnson, 1893; and 
other authors), and of Boveria (Pickard, 1927); (4) Conductivity, 
neuromotor system of many ciliates (Sharp, 1914; Yocom, 1918; and 
other authors). 

A fifth function, "metabolic influence," not previously noted, has re- 
cently been proposed by Parker (1929) for the fibrillar complex in 
Paramecium (Rees, 1922) and in other ciliates, comparable to the func- 
tion of fibrils in nerve cells. The neurofibrillar hypothesis for conduc- 
tivity in nerves was regarded as untenable by Parker (1929). After an 
extensive review of the evidences for and against this thesis, including 
Bethe's (1897) experiment on the brain neurones in the crab Carcinus, 
which showed that the nerve impulses did not have to traverse the fibrils 



CILIATE FIBRILLAR SYSTEMS 259 

of the cell body, Parker suggested that neurofibrils generally, and possi- 
bly the fibrils described for certain ciliates, may serve to transmit, from 
the metabolic center or nucleus, metabolic influences "essential for the 
continued life of the whole neurone." How these transmissions might 
be made was not clear. He thought they might involve "chains of ionic 
readjustment such as have been proposed as an explanation of the nerve 
impulse." Aside from whether or not such might apply to the function 
of the fibrils in ciliates, however, he rightly observed that these fibrils 
may not be intimately associated with the nucleus, as seems to be the 
case in neurones. 

Entz's (1893) interpretation of an elastic function for the Spasmonem 
in the recoiling stalk of the vorticellids was discussed under the caption 
"Interpretations." In addition to this, reference may be made briefly 
to Koltzoff's (1903, 1906, 1912) similar interpretations for elastic 
fibrils in cilia and in cells generally. He would ascribe elasticity to all 
fibrils in maintaining all organic form other than spherical. Since proto- 
plasm is liquid, as shown by the sphericity of its enclosed vacuoles, then 
elastic elements must be postulated to counteract the physical forces of 
inner and outer osmotic pressure and surface tension, which tend always 
to efl^ect spherical form. Such elements are fibrillar, as observed in 
the many kinds of cells investigated. The amount of evidence adduced 
by Koltzoff is impressive, but his interpretation obviously cannot apply 
exclusively to all fibrils. 

Similar claims for a supporting function for fibrils are rather wide- 
spread in the literature. Thus Jacobson (1931), as already stated, is 
disposed to attribute a supporting function to all noncontractile fibrils. 

These few citations, together with many others previously noted, may 
serve to indicate the diversity of functions that have been variously 
attributed to fibrillar differentiations in ciliates. In so far as they suggest 
that these fibrils and fibrillar systems may differ in their structure, func- 
tions, and relationships among the manifold kinds of ciliated Protista, 
certainly no one could present conclusive evidence to the contrary. But 
when, in the absence of proof, an investigator seriously contends that 
in these unicellular organisms any and all fibrillar differentiations per- 
form only one elementary function, whether it be that of elasticity, 
mechanical support, contractility, conductivity, or "metabolic transmis- 
sion," or when he assigns to these fibrils or fibrillar systems one or two 



260 CILIATE FIBRILLAR SYSTEMS 

functions to the exclusion of another possible function or functions, then 
surely that investigator thereby adopts a point of view which is incon- 
sistent and indefensible as well. 

In such instances we begin to sense a recrudescence of the opposing 
claims advanced in the Ehrenberg-Dujardin controversy and of the non- 
cellular theory of protistan organization proposed by Dobell and others. 
Once having denied the validity of Ehrenberg's extreme contention that 
the organs of the Infusoria are essentially miniature counterparts of 
those of macroscopic organisms, a comparably extreme viewpoint is sub- 
stituted, which would maintain that the Protista represent a complete de- 
parture in the organization of living things and so belong in the wholly 
exclusive category of non-cellular organisms. Thus the claims of these 
counter extremists would have us search for identities in organization, on 
the one hand, or only for differences in protistan and multicellular or- 
ganization on the other hand. 

In Ehrenberg's day similar extreme points of view were quite irrec- 
oncilable, but in our day they can scarcely represent anything less than 
rash inconsistencies. Obviously the thesis of non-cellular organization 
tends to place exclusive emphasis on differences between protozoan and 
metazoan organization and, if one is still inclined to accept that thesis, 
one might well refer to Belaf's (1926) excellent monograph on the 
protistan nucleus. Variable as are the nuclei, in form and behavior, of 
the many kinds there described and illustrated — where they appear to 
diifer from one another more than some differ from metazoan nuclei — 
surely one cannot fail to recognize that their numerous modifications do 
not represent discrete differences, but clearly betray the indelible marks 
of a common origin. They are like the musical variations of some great 
motif. They demonstrate irrefutably that living nature has been both 
labile and stable in its evolutionary history, so that we are amply justified 
in searching out and emphasizing not merely differences but also, and 
more fundamentally, similarities, in both the structural and functional 
processes of protoplasmic differentiation. 

And since all cellular differentiation is referable in its last analysis to 
protoplasmic differentiation, then certainly the fibrils and fibrillar sys- 
tems of multicellular tissues, such as those described by Grave and 
Schmitt (1925), may belong in the same category, both structurally and 
functionally, as some fibrillar differentiations that have been described 
and some that we may afford further to search for, in unicellular organ- 



CILIATE FIBRILLAR SYSTEMS 261 

isms. Knowing today the general properties and behavior of the long- 
chain protein molecules, if such fibrils are proteinaceous, as evidently 
they are, then fibrillar differentiation is one of the most likely kinds of 
protoplasmic differentiation that might be expected. 

But by the same token, we would not expect all such proteinaceous 
fibrils to be alike, either structurally or functionally. Both by virtue of 
their intrinsic properties and their relations to other organelles, some 
fibrils of protistan cells, or of metazoan cells, may serve for support, 
others for contraction, and still others for conduction of impulses to and 
from motor or other organelles. Or any one fibrillar complex may per- 
form more than one of these, or of other yet unkown, functions. And 
this duality or plurality of fibrillar functions may obtain for protistan 
cells and for tissue cells of multicellular organisms. Certainly we know 
of no evidences contradicting this possibility. 

The actual function or functions of most fibrils or fibrillar systems 
are not as yet finally known. There can be no doubt about the contractile 
properties of the myonemes of Stentor, and some experimental evidences 
indicate a coordinating (conductive) function for some fibrils in several 
ciliates and in epithelial tissue. The outer fibrillar systems of Varcimecium 
and other ciliates may be fibrillar, or only apparently fibrillar, as an 
integral part and pattern of the pellicle. If of the pellicle, then at least 
one of its functions would evidently be that of support. 

Much more study and more critical analysis of these fibrillar systems 
are greatly needed by both improved old and newly devised observational 
methods, perhaps such as that of the recently developed electron micro- 
scope. Then complementing these observational and comparative studies 
will be required indispensably, as the crucial test of all of our hypotheses, 
exceedingly refined and precise tools and methods of experimentation. 
Even today there are many devices suitable for this purpose, if properly 
adapted and fully utilized by the ingenious well-trained hands and eyes 
of thoroughly informed, exceptionally endowed minds. It is a mistake 
to suppose that such microtechniques are peculiarly difficult and that such 
problems are really unapproachable. It is rather that these techniques are 
different and that their use requires special training. With such training, 
it may be easier to transect a ciliate or a marine ovum, with much more 
accuracy, than to perform "free hand" some of the disections on macro- 
scopic organisms. 

By micromanipulative methods and with the aid of other modern 



262 CILIATE FIBRILLAR SYSTEMS 

devices, such as the ultracentrifuge, and by micromethods of irradiation, 
and the like, we may expect for the future, once the world has recovered 
its sanity, notable advances in protistological investigations such as may 
not have been dreamed of, even by the most sanguine of our predecessors. 
No other group of organisms may offer more than the unicellular forms 
toward the solution of some of our most fundamental problems. The re- 
sults, therefore, will provide a better understanding not only of these 
unicellular organisms, but of all forms of life. In their last analysis, how- 
ever, all such problems must surely depend for any final solution upon 
unique exacting methods of biological experimentation. ""Belief uncon- 
firmed by experiment is vain" (Francesco Redi, of Florence, 1668). 

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Anat. Physiol., 1864: 291-302. 

Meyer, A. 1920. Analyse dcr Zelle. Jena. 

Nercshcimer, E. R. 1903. Die Hohe histologischcr Differenzierung bei hetcro- 

trichcn Ciliaten. Arch. Protistenk., 2: 305-24. 

1907. Nochmals iiber Stentor coertdeus. Arch. Protistenk., 9: 137-38. 

Parker, G. H. 1929- What are neurofibrils.^ Amer. Nat., 63: 97-117. 
Pensa, A. 1926. Particolarita strutturale di alcuni protozoi cigliati in rapporto 

con la contratilia. Monit. zool. itaL, 37: 165-73. 
Pcschkowsky, L. 1927. Skelettgebilde bei Infusoricn. Arch. Protistenk., 57: 

31-57. 



CILIATE FIBRILLAR SYSTEMS 269 

Pickard, E. A. 1927. The neuromotor apparatus of Boveria ternedinidi Nelson, 

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405-28. 
Powers, P. B. A. 1933. Studies on the cihates from sea urchins. II. Entodiscus 

borealis (Hentschel). Behavior and morphology. Biol. Bull., 65: 122-36. 
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270 CILIATE FIBRILLAR SYSTEMS 

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53-65. 



CHAPTER V 

MOTOR RESPONSE IN UNICELLULAR ANIMALS 

S. O. Mast 

Responses consist of changes in structure, composition, form, or move- 
ment in organisms, which, in turn, are correlated with changes in the 
constituents of the environment or the organisms. Responses are found 
in all living systems and are among the most fundamental distinguish- 
ing characteristics of life. Motor responses consist of changes in rate 
or direction of movement of organisms or their constituents. They 
facilitate the control of the environment by the organisms involved and 
are consequently of great importance to them. Knowledge concerning 
these responses and their relation to the factors correlated with them 
makes it possible to control the activities of organisms, it throws light 
on the distribution of pain and pleasure (consciousness) which pro- 
foundly aifects the attitude of man toward his fellow creatures, and it 
illuminates the processes involved in instincts and in learning. Such 
knowledge is therefore very valuable. 

The unicellular organisms are in many respects extraordinarily favor- 
able for the study of the more fundamental characteristics of these re- 
sponses. They are relatively simple in structure. Many of them can be 
readily procured and maintained in great numbers, and the factors in 
their environment can be accurately controlled or changed as desired; 
and, in addition, details concerning the responses can be readily seen. 
Moreover, the motor responses in these organisms are very favorable 
for the study of adaptation, as pointed out long ago by Jennings (1906) . 

In the following pages are presented the more important facts in hand 
concerning the motor responses of the rhizopods, flagellates, ciliates, and 
colonial forms to light, electricity, and chemicals. These are presented 
with the view of encouraging the use of these organisms in further work 
on various biological problems. Important results have also been ob- 
tained on responses to contact, temperature, and gravity, but limitation in 
space prevents the consideration of these. 



272 



MOTOR RESPONSES 



Responses to Light 



A. RHIZOPODS 



Many of the rhizopods respond to light and some orient if they are 
exposed in a beam of light. Some of the responses are correlated with 
the rate of change in intensity, others are not. They are fundamentally 
the same in all the species which have been investigated, but they have 
been more thoroughly studied in Amoeba proteus than in any of the 
other species. The following considerations therefore refer largely to 
this species. 

A. proteus consists of a thin elastic outer membrane, the plasmalemma, 
a central relatively fluid granular mass, the plasmasol, surrounded by a 




G 



- 0" ' w 




Figure 93. Camera sketch of horizontal optical section of Amoeba proteus. Ps, plas- 
masol ; Pg, plasmagel ; PI, plasmalemma; HC, hyaline cap; Pgs, plasmagel sheet; L, 
Liquid layer; S, region of solation; G, region of gelation. (After Mast, 1926.) 



relatively solid granular layer, the plasmagel, and a thin fluid hyaline 
layer between the plasmagel and the plasmalemma (Fig. 93). During 
locomotion, the plasmalemma is attached to the substratum and to the 
adjoining plasmagel; the plasmagel at the posterior end is transformed 
into plasmasol, which flows forward to the anterior end and is there 
transformed into plasmagel. The forward flow of the plasmasol is due 
to contraction of the plasmagel at the posterior end and expansion at the 
anterior end, owing to difference in its elastic strength in these two re- 
gions. In some species surface tension is probably also involved in locomo- 



MOTOR RESPONSES 273 

tion. In A. mira, for example, the anterior portion consists of a thin 
sheet of hyaline cytoplasm in close contact with the substratum (Hopkins, 
1938). The surface tension at the water-substrate interface is probably 
greater than the combined surface tensions at the cytoplasm-substrate and 
cytoplasm-water interfaces. This would result in a spreading of the cyto- 
plasm over the substrate (like oil over water) wherever the two are in 
contact, i.e., at the anterior end of the organism. 

With reference to a marine amoeba, Pantin (1923-31) contends that 
the cytoplasm at the anterior end swells and extracts water "jrom the 
posterior protoplasm of the amoeba itself, and that this will cause a 
stream from the posterior to the anterior end." Presumably he holds that 
the cytoplasm shrinks there and gives it off. However, that would neces- 
sitate absorption of water during gelation at the anterior end, and elimina- 
tion during solation at the posterior end, which is contrary to what is 
ordinarily observed in the process of gelation and solation of gels. 

Marsland and Brown (1936) suggest that the forward flow is due to 
increase in volume during solation at the posterior end, and decrease 
in volume during gelation at the anterior end. They give no direct evi- 
dence in support of this suggestion, but simply say that "The magnitude 
of these volume changes in relation to the observed rate of flow is prob- 
lematical." 

Responses of Amoeba to light are therefore probably due to localized 
changes in (1) the elastic strength of the plasmagel, (2) the rate of 
transformation of plasmasol to plasmagel and vice versa, or (3) the 
firmness, extent, or region of attachment to the substratum (Mast, 1923, 
1926a, 1931a). 

Shock-reactions. — Engelmann (1879) long ago observed that if 
strong light is flashed on an amoeba, movement stops suddenly, but that 
if the intensity is gradually increased, movement continues. This response 
therefore depends upon the rate of change of light intensity. Such re- 
sponses are usually designated "Schreckbewegungen," or shock-reactions. 
They are closely correlated with adaptation. The shock-reaction in 
Amoeba produced by light varies greatly. It may consist merely of mo- 
mentary retardation in streaming in a localized region in a pseudopod, of 
total cessation throughout the entire animal with reversal in direction of 
streaming after recovery, or of any one of an endless number of modifica- 



274 



MOTOR RESPONSES 



tions between these extremes. The character of the response is correlated 
with the amount of Hght received, as well as with the rate of reception. 
There is no fixed threshold and the "all-or-none" law does not apply 
(Mast, 1931a). 

If an amoeba is intensely illuminated for a very short time only, move- 
ment does not cease until some time after the light has been cut off. 
The period between the beginning of illumination and the response is 



^ 



i 
2 
1 
C 



<^ 



Figure 94. Curves showing for Amoeba proteus the 
relation between luminous intensity, reaction time 
(RT), stimulation period (SP) and latent period (LP), 
and a hyperbola (H). Note that the curve for re- 
action time simulates a hyperbola, but that the curve 
for the stimulation period does not. This shows that 
the amount of light energy necessary for response 
varies with the intensity and that the Bunsen-Roscoe 
law does not apply. (Modified after Folger, 1925.) 



K 




a-T- 



lCMINOIJs INtENSITt 



-n rr 



known as the "reaction time"; the time illumination must continue, the 
"stimulation period"; and the time it need not continue, the "latent 
period" (Folger, 1925). There are therefore two processes involved in 
producing this response. The first occurs only in light, the other in light 
or in darkness. The action of light probably results in the formation of 
a substance which acts to produce, independent of light, another sub- 
stance which induces the response. 



MOTOR RESPONSES 275 

After an amoeba has responded to rapid increase in illumination, 
some time must elapse before it will respond again to the same increase 
in illumination. There is therefore a refractory period, a period during 
which the amoeba recovers from the effect of the stimulation. During 
a part of this period the amoeba may remain either in light of the same 
intensity, such as that which induced the response, in light of lower 
intensity, or in darkness; but during the remainder of the period it must 
be in light of lower intensity or in darkness. There are therefore two 
processes which occur during the refractory period, one ( 1 to 2 minutes) 
which proceeds with or without any change in luminous intensity, and 
another (10 to 20 seconds) which proceeds only if the intensity is de- 
creased. These processes result in the production of the physiological state 
which existed before the exposure; that is, in recovery (Folger, 1925). 

The latent period and the amount of light energy required to induce 
cessation of movement vary with the intensity of the light used (Fig. 94) . 
Figure 94 shows that as the intensity increases, the latent period increases 
rapidly from about one second at 500 ± meter-candles to a maximum 
of about 6 seconds at 1,000 rh meter-candles, and then decreases gradu- 
ally to about 0.75 seconds at 11,000 d= meter-candles; and that the light 
energy required to induce cessation of movement decreases from about 
7,000 Ht meter-candle seconds at 500 ± meter-candles to a maximum of 
about 24,000 ± meter-candle seconds at 1,500 dz meter-candles, and 
then increases to about 30,000 ± meter-candle seconds at 11,000 ± 
meter-candles. These results are, however, only rather crude approxima- 
tions. They were obtained by a method of calculation which yields results 
with a large probable error and they have not been confirmed. The data 
are, however, sufficiently accurate to substantiate Folger' s conclusion 
that the Bunsen-Roscoe law does not hold. 

This work should be repeated, and the latent period established by 
direct observation in all luminous intensities, instead of by calculation. 
This is especially desirable since recent experience makes it possible to 
select specimens of A. pro feus in which the responses are much more 
consistent than they were in those used by Folger. 

No explanation has been offered for the mode of variation in the 
latent period, with variations in luminous intensity during the period of 
stimulation. However, it has been suggested that the variation in the 



276 



MOTOR RESPONSES 



amount of light energy required to induce cessation of movement is due, 
at least in part, to adaptation (Mast, 1931a). For if the light is rapidly 
increased and then held, streaming soon begins again, i.e., the organism 
recovers from the effect of the increase in light. In other words, it be- 
comes adapted (Mast, 1939; Folger, 1925). This shows that the effect 
of rapid increase in light is eliminated while the organism is continuously 






Figure 95. Camera drawings of Amoeba sp. illustrating the response to localized 
illumination. Rectangular areas, regions of high illumination ; arrows, direction of proto- 
plasmic streaming; dotted lines in B, C and D, positions and forms shortly after the 
illumination of the parts indicated ; n, nucleus ; r, contractile vacuole. E and F, same 
specimen ; F, form and direction of streaming assumed by E after the anterior end had 
been illuminated for a few minutes. (After Mast, 1932.) 



exposed to the light. It also indicates that there are two opposing processes 
involved, i.e., that increase in light induces certain changes in the or- 
ganism and that internal factors tend continuously to oppose and to 
eliminate these changes. If this is true, the more rapidly a given amount 
of light is received, the less time there is for recovery, and consequently 
the greater will be the effect of a given quantity of light. This probably 
accounts for the increase in the amount of light energy required (with 
decrease in intensity) when observations are made in weak light; but it 



MOTOR RESPONSES 277 

does not account for the increase in the amount required (with increase 
in intensity) if the observations are made in strong light. 

The quantity of Hght energy required to induce cessation of movement 
depends upon the chemical composition of the surrounding medium. In- 
crease in HCL, for example, causes an increase in the quantity of light 
required. On the other hand, an increase in CO, causes decrease in the 
quantity required. In solutions of KCl, CaCl,, and MgClg, respectively, 
the quantity of energy required increases as the salt concentration de- 
creases, but in solutions of NaCl there is no consistent correlation be- 
tween the quantity of energy and the concentration of the salt. In gen- 
eral, the quantity of energy required appears to vary directly with the 
viscosity of the cytoplasm (Mast and Hulpieu, 1930). The observations 
made by these authors extended, however, over only a very limited range 
of environmental variation. The conclusions reached are therefore not 
applicable to wide ranges of variations in the environment (Mast and 
Prosser, 1932). 

Increase in the illumination of any localized region of an amoeba re- 
sults in an increase of the thickness of the plasmagel in this region (Fig. 
95). Increase in the illumination of the entire amoeba results in an 
increase in the thickness of the plasmagel at the tip of the advancing 
pseudopods. In turn, this causes a cessation of movement (shock-reac- 
tion). 

The shorter waves of light are more efficient in inducing this response 
than the longer waves (Harrington and Teaming, 1900; Mast, 1910). 
According to Inman, Bovie, and Barr (1926), ultra-violet light is prob- 
ably more efficient than visible light. Although the distribution in the 
spectrum of stimulating efficiency has not been precisely ascertained, 
Folger (1925) maintains that it is not closely correlated with tempera- 
ture. He did not thoroughly investigate the problem, however. 

Kinetic responses. — If an amoeba is kept for some time in very weak 
light it becomes inactive; if the light is then increased, the organism 
gradually becomes active again. This response is similar to the response 
to change in temperature. It is primarily correlated with the magnitude 
of the change, not with the rate of change in intensity. It is probably due 
to the effect of light on the rate of transformation of gel to sol and vice 
versa. This type of response occurs also in Difjlugia (Mast, 1931c), but 




22.5 7.5 15 

Time in light in minutes 

Figure 96. Relation between adaptation to light of different intensities and rate of 
locomotion in Amoeba proteus. Each point in the figure represents the average for one 
measurement on each of from fourteen to twenty-three specimens. The time in light 
is the time from the beginning of movement after exposure until the measurements were 
made. (After Mast and Stabler, 1937.) 



MOTOR RESPONSES 279 

the observations on it should be repeated and extended under carefully 
controlled conditions. 

Mast and Stabler (1937) made a thorough study of the relation be- 
tween luminous intensity and rate of locomotion in A. pro feus. They 
found that if dark-adapted amoebae are exposed to light, the rate of 
locomotion gradually increases to a maximum and then remains con- 
stant; that the time required to reach the maximum decreases from 15 
minutes at 225 meter-candles to a minimum of 7 minutes at 15,000 
meter-candles, and then increases to 30 minutes at 40,000 meter-candles; 
and that the rate of locomotion at the maximum increases from 128.8 zh 
10.8 micra per minute at 50 meter-candles to 219-3 ± 11-4 micra per 
minute at 15,000 meter-candles, and then decreases to 150.2 ±: 8.5 
micra per minute at 40,000 meter-candles (Fig. 96). They present evi- 
dence which indicates that the increase in rate of locomotion with in- 
crease in light intensity is due to the action of the longer waves, and that 
the decrease in rate in intensities beyond the optimum is due to the action 
of the shorter waves. This action of light on rate of locomotion is similar 
to the action of temperature. It is probably due to changes in the rate of 
sol-gel and gel-sol transformations. If this is true, both of these trans- 
formations must be augmented by the longer waves and retarded by the 
shorter. 

Orientation. — Davenport (1897) found that A. proteus orients fairly 
precisely in a beam of direct sunlight and that it is photonegative, but 
he did not ascertain the processes involved in orientation. 

Mast (1910) demonstrated that if an amoeba is unilaterally illu- 
minated, pseudopods develop more freely on the shaded side than on 
the illuminated side, and that this results in gradual turning from the 
light (Fig. 97). He concludes that orientation is due to retardation in 
the formation of pseudopods on the more highly illuminated side, owing 
to increase in the thickness of the plasmagel on this side caused by the 
gelating effect of light. 

There is some evidence which indicates that A. proteus is photopositive 
in very weak light (Schaeffer, 1917; Mast, 1931a). More carefully con- 
trolled observations concerning this are highly desirable. 

It is possible that the kinetic responses in Amoeba are due to changes 
in the rate of sol-gel transformations at the anterior end, and gel-sol 



280 



MOTOR RESPONSES 



transformations at the posterior end. Moreover, the shock reactions ap- 
pear to be associated with rapid local increases in the sol-gel transforma- 
tion. If the views concerning the process of orientation as presented 




Figure 97. Camera outlines representing different stages in the process of orientation 
in Amoeba protens. 1, Amoeba oriented in light /'; 2-9, successive positions after ex- 
posure to light /, time indicated in each. Arrows represent the direction of streaming 
of protoplasm in the pseudopods. In those which do not contain arrows there was no 
perceptible streaming at the time the sketch was made. / and /' direction of light; mm, 
projected scale. (After Mast, 1910.) 

above are correct, it is obvious that orientation is the result of shock 
reactions rather than kinetic reactions. 



B. FLAGELLATES 

Response to rapid changes in the intensity of light is very widespread 
among the flagellates, and many of them orient fairly precisely. The 
processes involved are essentially the same in all. Euglena is representa- 
tive of those which orient, and Peranema trkhophorum is representative 
of those which do not. 

Euglena rotates continuously on its longitudinal axis as it swims. The 
flagellum extends backward along the ventral or abeyespot surface. This 
causes continuous deflection of the anterior end toward the opposite 



MOTOR RESPONSES 



281 



surface, resulting in a spiral course. Its direction of movement is changed 
by the shifting of the distal end of the flagellum from the surface of the 
body so as to increase the angle between it and the surface. This in- 
creases the deflection of the anterior end (Fig. 98). 

Shock reaction and aggregation. — Engelmann (1882) observed that if 
the intensity of the light in a field in which euglenae are swimming about 
at random is rapidly decreased, they stop suddenly, then turn and pro- 



—'^nf] 



A B 

Figure 98. A. Diagrams showing the position of the flagellum as seen in a viscid 
medium; a, when Euglena is swimming forward in a narrow spiral; b, when swerving 
sharply towards the dorsal side; c, when moving backwards. B. Dotted area, shows the 
position of the moving India-ink particles, a, when Euglena is swimming forward in a 
narrow spiral ; b, when swerving toward the dorsal side during a shock-movement. 
(After Bancroft, 1913.) 



ceed in various directions. He designated the response as a "Schreck- 
bewegung" (fright movement, or shock reaction), because the re-ori- 
ented organisms gave the impression of having been frightened. It was 
found that if the intensity is slowly changed this response does not 
occur. It is therefore dependent upon the rate of change in intensity. 

He says that if there is a spot of relatively strong light in the field, 
it acts just like a trap; owing to random movements, the euglenae get 
into this spot, but as they reach the boundary on the way out, the rapid 
reduction in intensity induces the shock reaction and consequently pre- 
vents their exit. 

Under some conditions the euglenae respond to rapid increase in in- 



282 MOTOR RESPONSES 

tensity and aggregate in a spot of relatively weak light in the field 
(Mast, 1911). 

Orientation. — If euglenae are exposed in a beam of light, they usually 
swim toward or away from the source of light, i.e., they may be either 
photopositive or photonegative. 

Verworn (1895) postulated that if the euglenae are not directed to- 
ward or away from the light, so that one side is more strongly illuminated 
than the other, the flagellum beats more effectively in one direction 
than in the opposite, that this causes the euglenae to turn until both sides 
are equally illuminated, and that the flagellum then beats equally in op- 
posite directions and the organism moves directly toward or away from 
the source of light. 

The above hypothesis is, in principle, essentially the same as that 
formulated by Ray (1693), in reference to orientation in plants, and 
later accepted by de Candolle (1832). According to this idea, the effect 
of light on the activity of the motor mechanism, or upon the photo- 
receptors connected with it, is dependent upon the intensity (not upon 
change of intensity) of the illumination. The light acts continuously 
after orientation has been attained, as well as during the process of 
orientation. During the process of orientation, the illumination on op- 
posite sides is unequal, which results in quantitatively unequal action 
in the motor mechanism; but after orientation, it is equal on opposite 
sides, and consequently the action of the mechanism on opposite sides 
is equalized. Verworn applied this theory to ciliates as well as flagellates. 
In his earlier work, Loeb (1890) strenuously opposed the theory out- 
lined above, accepting Sachs's "ray-direction theory" as the alternative. 
He adopted it later (1906), however, and applied it to higher animals, 
introducing the idea that the action of the locomotor appendages is 
quantitatively proportional to the intensity of the light on the photo- 
receptors connected with them. He maintained that this is due to the 
effect of light on muscle tonus. This theory has been designated the 
"difference of intensity theory," the "continuous-action theory," the 
"tropism theory," and "Loeb's muscle-tonus theory" (Mast, 1923). 

Engelmann (1882) demonstrated that only the anterior end of 
Euglena is sensitive to changes in luminous intensity. Jennings (1904) 
contends that because of this, all turning from the light results in a 
reduction of illumination, whereas all turning toward the light results 



MOTOR RESPONSES 283 

in an increase in illumination of photosensitive substance. The photoposi- 
tive specimens consequently turn until they face the light, whereas photo- 
negative specimens turn until they face in the opposite direction. When 
the stimulus which induces turning ceases, the organisms continue either 
directly toward or from the light. 

Mast (1911) made a very intensive study of the process of orientation 
in a species of Euglena which crawls on the substratum but continuously 
rotates on the longitudinal axis as it proceeds. This Euglena orients very 
precisely in light, it has a well-developed eyespot, and it moves so slowly 
that the different phases of its responses can readily be followed in 
detail. It is therefore very favorable for the study of the process of 
orientation. 

If the intensity of the light is rapidly decreased in a beam in which 
specimens are proceeding toward the source of light, they stop suddenly 
and bend in the middle toward the abeyespot surface until the two halves 
form nearly a right angle; then they begin again to rotate on the longi- 
tudinal axis; and, while rotating, they gradually straighten and proceed 
once more toward the light source. If the intensity of the light is in- 
creased, or if it is slowly decreased, there is no perceptible response. The 
cessation of movement and the bending are therefore dependent upon 
the rate of decrease in the intensity of the light in the field, i.e., it is a 
shock reaction. The decrease in the intensity of light in the field neces- 
sarily results in decrease in intensity of light on all the substance in the 
field; it therefore must cause decrease in the illumination of the photo- 
sensitive substance. The response, then, is dependent upon the rate of 
decrease in the light on the photosensitive substance. 

If the direction of the beam of light is changed through 90^ without 
alteration in intensity, the specimens oriented in it are illuminated 
laterally. Those in which the eyespot surface faces the light after the 
direction of the rays has been changed, stop at once. They bend in the 
middle toward the abeyespot surface, then rotate, and gradually 
straighten to resume their crawling movements. Those in which the eye- 
spot surface does not face the light after the direction of the rays has been 
changed, do not respond to the changed direction of the rays, until, in 
the process of rotation on the longitudinal axis, this surface faces the 
light; then they also stop, bend, rotate, straighten, and proceed. Thus 
they continue until, in the process of rotation, the eyespot surface again 



284 



MOTOR RESPONSES 



faces the light, when they again respond. The gradual straightening 
during rotation results in greater deflection of the anterior end toward, 




■^^ 



Figure 99. Euglena sp. in a crawling state, showing details in the process of orienta- 
tion; p, contractile vacuole; es, eyespot; n, o. direction of light; a-c, positions of 
Euglena with light from n is intercepted ; c-m, positions after light from n is turned 
on and that from o cut off, so as to change the direction of the rays. If the ray direction 
is changed when the Euglena is in position c, there is no reaction until it reaches d. 
Then it suddenly reacts by bending away from the source of light to e. after which 
it continues to rotate and reaches position /, where it gradually straightens to g, and 
rotates to h, when the eyespot again faces the light and the organism is again stimulated 
and bends to /, from which it proceeds to /, and so forth. If the ray direction is changed 
when the Euglena is at d, it responds at once and orients as described above. If the 
intensity from n is lower than that from o the organism may respond at once when the 
ray direction is changed, no matter in which position it is. (After Mast, 1911.) 

rather than away from the light. Thus the anterior end becomes directed 
more and more nearly toward the light source, until an axial position 
is reached in which changes in illumination of the eyespot surface, owing 



MOTOR RESPONSES 



?85 



to rotation, disappear. The organism is then oriented (Fig. 99). The 
response induced by changing the direction of the rays, or by rotation in 
lateral illumination of uniform intensity, is precisely the same as the 
response induced by a decrease in the intensity of the light in the field 




a.'s. 



- -iC.U. 



Figure 100. Side view of anterior end of Euglena viridis. e, pigmented portion of 
eyespot; /, ilagellum; e.f, enlargement in flagelium; c.v, contractile vacuole; e.s. eye- 
spot surface of the organism; a.s. abeyespot surface of the organism. (After Wager, 
1900.) 

without a change in the surface illuminated. The change from illumina- 
tion of the anterior end or the abeyespot surface to illumination of the 
eyespot surface therefore must, in some way, result in a rapid decrease 
in the illumination of the photosensitive substance. How is this brought 
about? 

Wager (1900) demonstrated that the eyespot in Euglena consists 
of a spoon-shaped portion containing red pigment and a small globular 
enlargement of one of the roots of the flagelium in the concavity of the 
pigmented portion (Fig. 100). The eyespot is situated near the eyespot 



286 MOTOR RESPONSES 

surface, a short distance from the anterior end, with the convex surface 
directed outward and backward. When the anterior end, or the abeyespot 
surface, is directed toward the hght, the enlargement in the eyespot is 
fully exposed; but when the eyespot surface faces the light, the enlarge- 
ment is in the shadow cast by the pigmented portion. It is evident, then, 
that rapid change from illumination of the anterior end, or the abeyespot 
surface, to illumination of the eyespot surface causes rapid decrease 
in illumination of the enlargement in the eyespot, and that if the en- 
largement is photosensitive, change in the direction of the rays or rota- 
tion on the longitudinal axis has the same effect as decrease in the in- 
tensity of the light in the field. It is therefore highly probable that the 
enlargement in the eyespot is photosensitive and that the pigmented por- 
tion functions in producing changes in intensity of light on it, when the 
axial position of the organism changes and when it rotates on the longi- 
tudinal axis in lateral illumination. This contention is supported by the 
facts that the region of maximum stimulating efficiency in the spectrum 
is in the blue for Euglena (Mast, 1917) and that blue is absorbed by the 
yellowish-red pigmented portion of the eyespot (Fig. 102). 

In photonegative specimens the responses to changes in light intensity 
in the field and to changes in the surfaces illuminated are precisely like 
those of photopositive specimens, except that the responses are induced 
by (1) increase rather than decrease in light intensity and (2) by change 
from illumination of the eyespot surface to illumination of the abeyespot 
surface. 

The process of orientation in free-swimming specimens is, in prin- 
ciple, precisely the same as it is in crawling specimens. 

Orientation in Euglena is, then, clearly due to a series of responses 
dependent upon the rate of change in the intensity of the light on the 
photosensitive substance, which is probably situated in the concave sur- 
face of the pigmented portion of the eyespot. The light does not act 
continuously, and there is no evidence whatever indicating anything in 
the nature of balanced or antagonistic action of locomotor appendages 
on opposite sides, in accord with the Ray-Verworn theory. 

The evidence in hand indicates, in short, that the photosensitive sub- 
stance is confined to the concavity in the pigmented portion of the eye- 
spot; that rotation on the longitudinal axis results in alternate shading 
and exposing of this substance, if the organisms are not directed toward 



MOTOR RESPONSES 287 

or from the light; that this induces shock reactions which result in 
orientation; and that the organisms remain oriented and proceed directly 
toward or away from the light, because, after they have attained either 
of these two axial positions, rotation no longer produces changes in the 
illumination of the photosensitive substance in the eyespot, and they 
therefore continue in the direction assumed. In other words, the orienting 
stimulus ceases after the organism has become oriented. The organism 
then continues directly toward or away from the light because (1) 
owing to internal factors, it tends to take a straight course, and because 
( 2 ) if for any reason it is turned from this course, the orienting stimulus 
immediately acts, and induces shock reactions which bring it back on 
its course. 

Bancroft (1913) presented evidence against the contention that 
photic orientation in Euglena is due to shock reactions and concluded 
that it is due to tonus effects brought about by "the continuous action 
of the light," in accord with his conception of Loeb's tropism theory. 
Mast (1914) demonstrated, however, that if the evidence presented 
by Bancroft is valid, it proves that his explanation of orientation in 
Euglena is not correct. Moreover, the fact that after Euglena is oriented, 
the rate of locomotion is practically independent of the luminous in- 
tensity (Mast and Cover, 1922) also militates against his explanation. 
Orientation in light frotn two sources. — In a field of light consisting 
of two horizontal beams crossing at right angles, Ei/glena orients and 
goes toward or away from a point between the two beams. The location 
of this point is related to the relative intensity of the two beams in such 
a way that the tangent of the angle between the direction of locomotion 
and the rays in the stronger beam is approximately equal to the intensity 
of the weaker divided by that of the stronger (Fig. 101) (Buder, 1917; 
Mast and Johnson, 1932). Buder maintains that this demonstrates that 
there is a quantitative proportionality between the stimulus and the re- 
sponse. Mast and Johnson conclude that "it has no bearing on the prob- 
lem concerning the quantitative relation between stimulus and response," 
but that it can be explained on the assumptions that the eyespot is a 
photoreceptor and that the stimulating efficiency of light varies with the 
angle of incidence. 

Wave length and stimulating efficiency. — The shorter waves in the 
visible spectrum are more efficient than the longer in stimulating Euglena 




25 JO 

Obser\/eaf 

Figure 101. Graphs showing the relation between the direction of locomotion ob- 
served in a field of light produced by two horizontal beams crossing at right angles 
and that demanded by the "Resultantengesetz." Abscissae, angles between the direction 
of locomotion and the direction of the rays in the stronger beam observed with dif- 
ferent ratios of intensities in the two beams, ranging from at 0° to 1 at 45° ; ordinates, 
angles between the direction of locomotion and the direction of the rays in the stronger 
beam demanded by the "Resultantengesetz." 3 Euglena rubra; O Gonium pectorale ; # 
Volvox minor; O Volvox globator. (After Mast, 1907.) Note that if the observed 
direction of locomotion were the same as the theoretical all the points would fall on 
the broken line, and that this practically obtains for Euglena but not for Volvox and 
Gonium. Note also that for the latter, as the ratio between intensity in the two beams 
decreases from 1, the difference between the theoretical and the observed results in- 
creases to a maximum, then decreases to zero, after which it increases in the opposite 
direction. (After Mast and Johnson, 1932.) 



MOTOR RESPONSES 



289 



and other flagellates. Strasburger (1878) concluded that stimulation is 
confined to violet, indigo, and blue in the solar spectrum, with the maxi- 
mum in the indigo. Engelmann (1882) maintains that for Euglena the 
maximum is in the blue between 470 m\x and 490 m^, and Loeb and 




Figure 102. Curves representing the distribution in the spectrum of stimulating 
efficiency, constructed from data given in Table 15 (See Mast 1917). A, Pandorina (nega- 
tive) ; B, Pandorina (positive); C, blowfly larvae; D, Euglena viridis (negative); E, 
Euglena viridis (positive) F, Euglena tripteris (negative) ; G, Avena sativa (oat seed- 
lings). (Constructed from data obtained by Blaauw.) The circles represent points experi- 
mentally established abscissae, wave lengths ; ordinates, relative stimulating efficiency on 
the basis of equal energy. The curves for Eudorina and Spondylomorum, not represented in 
the figure, are in position and form essentially like those for Pandorina; the curve for 
Chlamydomonas is much like that for blowfly larvae; those for Euglena gracilis, E. 
minima, E. granulata, Phacus, Trachelomonas, Gonium, Arenicola, and Lumbricus are 
nearly like those for E. viridis and E. tripteris. (After Mast, 1917.) 

Maxwell (1910) assert that in the carbon-arc spectrum it is between 460 
and 510 m\i. The unequal distribution of energy in the spectrum was not 
considered in these conclusions. Mast (1917) made corrections for un- 
equal distribution of energy and ascertained the relative stimulating effi- 
ciency of negative and positive orientation at intervals of 10 )?i\\ through- 



290 MOTOR RESPONSES 

out the visible spectrum. He found that as the wave length increases, 
the stimulating efficiency also increases very rapidly from zero at about 
410 wp to a maximum of 21 arbitrary units at 485 wp, and then de- 
creases equally rapidly to zero at about 540 wp (Fig. 102). He holds, 
however, that the limits of the stimulating region depend upon the 
luminous intensity. 

Kinetic responses. — If Euglena is subjected for long periods to low 
illumination or to darkness, it gradually becomes less active; and if the 
illumination is then increased, it gradually becomes more active. The 
rate of change in activity varies with the magnitude of the change in 
intensity. But this response is never so sudden and abrupt as the shock 
reaction. There are therefore two types of responses to light in Euglena, 
one depending primarily upon the rate of change in luminous intensity, 
the other primarily upon change in the amount of light received. The one 
results in orientation and aggregation, the other in change in activity. 

Mast and Gover (1922) measured the rate of locomotion in several 
different flagellates in different intensities of light and found very little 
correlation between rate and intensity. The environmental factors were, 
however, not accurately controlled, and adaptation was not considered. 
The measurements should therefore be repeated, with the methods used 
by Mast and Stabler (1937) in their observations on Amoeba. 

Reversal in response. — Euglena is ordinarily photopositive in weak 
light and photonegative in strong light. The orienting response therefore 
tends to keep it in light of moderate intensity, indicating that these re- 
sponses are fundamentally adaptive. This has not been demonstrated, 
however, because the direction of orientation is not specifically correlated 
with luminous intensity. For example, euglenae which are strongly photo- 
positive in a given intensity of light at room temperature may become 
equally strongly photonegative if the temperature is rapidly decreased 
10 to 15 degrees, the extent of the requisite decrease depending upon 
the state of adaptation (Mast, 1911). 

This problem is much in need of thorough investigation. It is a very 
important problem because it concerns the biological significance of 
response to light in these organisms. 

II. Peranema tricophorum. — Peranema is a colorless flagellate with- 
out an eyespot. It is usually in contact with the substratum and moves 
slowly with the flagellum extending forward (Fig. 103). If the luminous 



MOTOR RESPONSES 



291 



intensity is rapidly increased, it stops suddenly and then deflects the 
anterior end sharply to one side. If the intensity is slowly increased, or if 
it is decreased, there is no response. The entire organism is sensitive to 




0.1 TTL jri. 



i 



Figure 103- Camera drawings illustrating the response of Peranema to contact or to 
rapid increase in luminous intensity. Al, normal locomotion ; 2, immediately after re- 
sponse; 3, 4, recovery from response; B, response to contact with grain of sand, o, and 
recovery. Note that response results in a change in the direction of motion of approxi- 
mately 90 degrees. (Mast, 1912.) 

light, but the flagellum is most sensitive and the posterior end least sen- 
sitive (Shettles, 1937). 

Dark adaptation. — Mast and Hawk (1936) demonstrated that if 
light-adapted peranemae are subjected to darkness, the time required in 
light of 2,000 meter-candles to induce the response decreases from 30.95 
seconds after 15 minutes in darkness to a minimum of 4.54 seconds after 
one hour in darkness, then increases to a maximum of 63.46 seconds 



292 



MOTOR RESPONSES 



after 6 hours in darkness, and then remains nearly constant. As the time 
in darkness increases, the sensitivity to hght rapidly increases to a maxi- 
mum, then decreases to a minimum, before it becomes constant (Fig. 
104). 



so 






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6 8/0 

TIME IN DARk^NCSS IN HOURS 



12 



14 



24 



Figure 104. Graph showing the effect of dark-adaptation on sensitivity to light in 
Peranema trichophorum. Each point on the curve, except the last two, represents the 
average reaction time for from fifteen to seventeen tests. (After Mast and Hawk, 1936.) 

Light adaptation. — Shettles (1937, 1938) made a much more exten- 
sive and thorough investigation of this response. He confirmed the con- 
clusions reached by Mast and Hawk (1936). A brief summary of other 
results obtained, and the conclusions reached, follows: 

If dark-adapted peranemae are subjected to light, their sensitivity to 
light increases rapidly to a maximum, then decreases considerably, after 
which it remains nearly constant (Fig 105). The reaction time consists 



MOTOR RESPONSES 



293 



of an exposure period and a latent period. With increase in intensity 
of illumination, the exposure period decreases, at first rapidly, then 
more slowly, until it becomes nearly constant; the latent period increases 
to a maximum and then decreases, and the amount of light energy re- 
quired during the exposure period increases from 22,970 meter-candle 
seconds at 538 meter-candles to 54,315 meter candle seconds at 2,152 
meter-candles, and then decreases to 13,498 meter-candle seconds at 



c 

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TIME. IN LIGHT IN H0UR5 

Figure 105. Graphs showing rate of light-adaptation. Dark-adapted peranemas were 
exposed to light (intensity given in the graph) for the time indicated, then subjected 
to darkness one half hour, then exposed to 2,152 m.c, and the reaction time measured. 
Each point on the curves represents the average reaction time for ten tests, different 
individuals being used in each test. (After Shettles, 1937.) 

6,458 meter-candles (Fig. 106) . He concludes that "the amount of light 
energy required to induce a shock-reaction in Peranema varies greatly 
with the intensity of the light and that the Bunsen-Roscoe law conse- 
quently does not hold." 

The latent period decreases from 39-68 seconds at 10° C. to 24.3 
seconds at 30° (15.38 seconds), but the exposure period decreases from 
27.87 to 22.97 seconds (only 5.8 seconds). This indicates that there 



MOTOR RESPONSES 




%53d lOli, ZI5Z 3229 430S S332 (^56 
LUMINOUS INTENSITY IN METER. CANDLES 



aui 



Figure 106. Graphs showing relation between luminous intensity and reaction time, 
exposure period, latent period, and energy. Each point on the curves of the reaction 
time and the exposure period represents the average for ten tests, different individuals 
being used in each test. Each point on the curve of the latent period represents the dif- 
ference between the reaction time and the exposure period. Each point on the energy 
curve represents exposure period X luminous intensity. (After Shettles, 1937.) 

are at least two processes involved in the response of Peranema to light. 
One of these is nearly independent of temperature and is therefore prob- 
ably photochemical. The other is closely correlated with temperature and 
is therefore not photochemical. 

After Peranema has responded in light of a given intensity, it must 
be subjected to light of a lower intensity or to darkness before it will 
again respond. The time required for recovery varies directly with the 



MOTOR RESPONSES 295 

luminous intensity in which the response occurred, and inversely with the 
temperature. 

Wt^ve length and stimulation. — Stimulating efficiency of light is 
closely correlated with wave length. There are two maxima in the spec- 
trum, one in the ultra-violet at 302 w^ and one in the visible at 505 W(j. 
The latter is nearly twice as great as the former. 

The absorption of light by Peranema in the violet remains nearly 
constant as the wave length decreases from 450 m\\ to 325 wp, then 
increases rapidly and extensively to a maximum at 253 »2p. The maxi- 
mum injuring efficiency is also at 253 m\!,. Injury is therefore closely 
correlated with the amount of light absorbed, but stimulating efficiency 
is not, for the maximum is at 302 }n\\ in place of 250 my\. The processes 
involved in stimulation therefore differ from those involved in injury. 
Injury, in Peranema, is due to coagulation of the protoplasm, whereas 
stimulation is probably not due either to coagulation or to increased 
viscosity. 

Peranema responds very precisely and very consistently. Its move- 
ments are very slow and its reaction time long. Since it can be grown 
under fairly accurately known environmental conditions in total dark- 
ness, it is well suited for quantitative work of a high order. 

C. CILIATES 

Very few of the ciliates respond to light. Only one of these, Stentor 
coeruleus, has been investigated extensively. 

If the luminous intensity is rapidly increased, this organism stops, 
turns toward the aboral surface, and then proceeds. This is a shock 
reaction, because if the intensity is slowly increased there is no response. 
If the intensity is decreased, no matter whether rapidly or slowly, there 
is no response. If Stentor is exposed in a beam of light, it orients fairly 
precisely and swims away from the light, i.e., it is photonegative. It 
rotates on the longitudinal axis as it swims, so that if it is not oriented, 
the oral and the aboral surfaces are alternately shaded and illuminated. 
The oral surface is much more sensitive than the aboral; therefore every 
time that this surface is carried from the shaded to the illuminated side, 
the result is the same as an increase in the illumination of the entire 
organism, and it consequently responds, i.e., it turns toward the aboral 
surface. This continues until it is directed away from the light, and 




Figure 107. Stentor coeruleus in the process of orientation. Curved line, spiral course; 
arrows m and n, direction of light from two sources ; a-j, different positions of Stentor on 
its course ; o, oral surface ; ab, aboral surface. At a the Stentor is oriented in light from 
m, n being shaded. If n is exposed and ?« shaded simultaneously when the Stentor is 
in position b, there is usually no reaction until it reaches c and the oral side faces the 
light; then the organism may respond by suddenly stopping, backing, and turning sharply 
toward the aboral side (dotted outline), and become oriented at once; or it may merely 
swerve toward the aboral side without stopping. At e the oral side is again exposed, 
and the organism is again stimulated and it again swerves from the source of light. 
This process continues until the oral side is approximately equally exposed to the light 
in all positions on the spiral course. If the Stentor is at c when n is exposed, it responds 
at once and orients as described above. If the light from n is more intense than that 
from m, or if the organism is very sensitive when n is exposed and w shaded, it re- 
sponds at once, no matter in which position it is. If it is at b, it turns toward the source 
of light, but now repeats the reaction, successively turning in various directions until 
it becomes oriented. (After Mast, 1911.) 



MOTOR RESPONSES 297 

rotation no longer produces changes of intensity on the opposite sur- 
faces (Fig. 107). Photic orientation in Stentor is therefore the result 
of a series of shock reactions, as is the case in Euglena. There is no evi- 
dence in support of the view that it is the result of a continuous quanti- 
tative difference in the activity of the cilia on opposite sides, in pro- 
portion to the difference in the illumination of these sides. The process 
of orientation in Stentor is therefore not in accord with the Ray-Verworn 
theory. 

If stentors are exposed in a field of diffuse, non-directive light which 
contains a dark spot, they aggregate in this spot. The process of ag- 
gregation is, in principle, precisely the same as the process of aggrega- 
tion of photonegative euglenae in a dark spot. They reach the dark spot 
by random movements. No reaction occurs when the organisms enter the 
unilluminated area. However, at the periphery on the way out, as the 
light intensity rapidly increases, they stop suddenly, turn sharply to- 
ward the aboral surface, and then proceed in a different direction. The 
dark spot therefore acts like a trap (Jennings, 1904; Mast, 1906, 1911) • 

The relative stimulating efficiency of different regions in the spectrum 
has not been investigated; no observations have been made on the 
quantitative relation between the different phases of the shock reaction, 
the state of adaptation, and the extent of change in luminous intensity. 
Indeed, very little is known about the body processes involved in stimula- 
tion and response. 

D. COLONIAL ORGANISMS 

Response to light is essentially the same in all of the colonial forms 
in which it has been studied, but it has been more intensively investigated 
in Volvox globator than in any of the other species. 

Volvox is a slightly elongated, globular colonial organism somewhat 
less than one mm. in diameter. It consists of numerous cells (zooids), 
each of which contains two flagella and an eyespot. The zooids are ar- 
ranged in a single layer at the surface of the colonies. The eyespot in each 
zooid is directed toward the posterior end of the colony, but those at the 
anterior end are much larger than the rest (Fig. 108). 

Mast (1927a) presented evidence which demonstrates that the eye- 
spots consist of a cup-shaped pigmented portion, a lens-like structure 
near the opening of the cup, and photosensitive substance between this 



298 MOTOR RESPONSES 

and the inner surface of the cup. The evidence also indicates that the 
lens-hke structure brings the longer incident waves of light to focus in the 
wall of the cup; and that the shorter wave lengths, after being reflected 
from the inner surface of the cup, are focused in the photosensitive sub- 
stance (Fig. 109). 



Figure 108. Camera drawing showing the zooids in about one half of an optical sec- 
tion through the longitudinal axis of a colony of Volvox. l-a. longitudinal axis of 
colony; a, anterior end; z, zooids, /, flagella; e, eyes. Note that the eyes are located at 
the outer posterior border of the zooids and that they become larger as the anterior 
end of the colony is approached. (After Mast, 1927.) 

Movement, response, and orientation in V . globator have been thor- 
oughly studied by Mast (1907, 1926b, 1927b, 1932b). The more im- 
portant of the results obtained in this study lead to the following con- 
clusions. 

Shock reaction. — Volvox colonies rotate on the longitudinal axis as 
they swim. This is due to the diagonal stroke of the flagella. In a beam 
of light they usually orient and go almost directly either toward or away 
from the light, i.e., they may be photopositive photonegative, or neutral. 

If, while the colonies are swimming toward the light, the intensity is 
rapidly decreased without any change in the direction of the rays, rota- 
tion on the longitudinal axis stops and forward movement increases 



MOTOR RESPONSES 



299 



greatly. On the other hand, if the intensity is rapidly increased, the for- 
ward movement stops and the rate of rotation increases. If the colonies 
are swimming away from the light, the reverse occurs, i.e., forward move- 
ment decreases if the intensity is increased, and increases if it is de- 
creased. If the colonies are neutral, there are no such responses to changes 
of intensity. These responses consist chiefly, if not entirely, of rapid 
changes in the direction of the stroke of the flagella. In other words, a 
rapid decrease in the illumination of photopositive colonies changes the 




Figure 109. Sketches showing the structure of the eyespot in Volvox and its action 
on light entering the pigment-cup at different angles, p, pigment-cup; /, lens; y, yel- 
low focal spot; b, bluish green focal spot; ss, photosensitive substance; large arrows, 
incident rays of light. Note that the longer waves of the incident light are brought to 
focus in the wall of the pigment-cup and that the shorter waves are brought to focus in 
the cup, after being reflected from the inner surface, and then continue in the form 
of a concentrated beam of bluish-green light. Note also that the more obliquely the 
incident light enters the pigment-cup, the nearer the edge of the cup the yellow focal 
spot is located. (After Mast, 1927.) 

stroke of the flagella from diagonally backward to straight backward. 
An increase in the illumination causes it to change from diagonally back- 
ward to sidewise. In photonegative colonies precisely the reverse obtains. 
These responses continue for only a few seconds, although if the luminous 
intensity is slowly changed they do not occur at all. They are therefore 
dependent upon the rate of change in intensity, i.e., they are shock- 
reactions which are somewhat similar to those observed in Euglena. 

Kinetic responses. — If Volvox is kept in weak illumination or in dark- 
ness for several hours, it becomes inactive; but if the illumination is 
afterwards increased, it gradually becomes active again. These responses 
consist chiefly, if not entirely, in changes in the rate or the efficiency of 



300 MOTOR RESPONSES 

the stroke. Changes in the direction of the stroke of the flagella are not 
involved. They are relatively slow responses which occur, even if the 
luminous intensity is gradually changed. The responses are primarily 
dependent upon change in luminous intensity, not upon the rate of 
change. Consequently, there are, in Volvox, two different types of re- 
sponse: (1) typical shock reactions, and (2) responses which consist 
merely in changes in activity. 

Holmes (1903) maintains there is no consistent correlation between 
luminous intensity and rate of locomotion in Volvox. But his methods 
did not exclude the effect of adaptation. Further work concerning this 
correlation is therefore highly desirable. 

Orientation. — If a colony of Volvox in a beam of light is laterally 
illuminated, it turns gradually until it is oriented, and then proceeds 
either toward or away from the light source. When it is laterally illumi- 
nated, the zooids, owing to rotation of the colony on the longitudinal 
axis, are continuously transferred from the light side to the dark side, and 
vice versa. As the zooids pass from the light side to the dark side, the 
photosensitive substance in the eyespots becomes shaded by the pigment 
cup. As they pass from the dark side to the light side, this substance be- 
comes fully exposed. A rapid decrease in the illumination of the sensitive 
substance on the dark side of photopositive colonies induces shock reac- 
tions on this side, and the flagellar stroke increases in its backward phase. 
A rapid increase in illumination of the sensitive substance on the light 
side of a colony induces shock reactions. The latter consist of increase in 
the lateral phase of the stroke of the flagella (Fig. 110). This difference 
in the direction of the stroke of the flagella causes the colonies to orient 
gradually, until they are directed toward the light, after which all sides 
are equally illuminated. Rotation on the longitudinal axis then no longer 
produces changes in the illumination of the photosensitive substance, and 
the shock reactions cease. The Volvox colonies continue directly toward 
the light because, in the absence of external stimulation, they tend to 
take a straight course. Furthermore, if they are forced out of their course, 
opposite sides immediately become unequally illuminated, the intensity 
of the illumination of the photosensitive substance in the eyespots 
changes, and consequently reorientation occurs. 

If photopositive colonies are exposed in a field of light consisting of 
two horizontal beams which cross at right angles, they orient and swim 
toward a point between the two beams. The location of this point de- 




Figure 110. Diagrammatic representation of the process of orientation in Volvox. 
A, B, C, D, four zooids at the anterior end of the colony; l-a, longitudinal axis; large 
arrows, direction of illumination ; small arrows, direction of locomotion ; curved arrows, 
direction of rotation ; /. flagella ; e, eyes, containing a pigment-cup represented by a 
heavy black line and photosensitive tissue in the concavity of the cup. Note that when 
the colony is laterally illuminated, the photosensitive tissue in the eyes on the side 
facing the light is fully exposed and the flagella on this side beat laterally. Those on the 
opposite side, shaded by the pigment-cup and the flagella on this side, beat directly 
backward. The difference in the direction of the beat of the flagella on these two sides is 
due to alternate decrease and increase in the luminous intensity to which the photo- 
sensitive tissue in the eyes is exposed, owing to the rotation of the colony on its longi- 
tudinal axis — an increase causing, in photopositive colonies, a change in the direction 
of the stroke of the flagella from backward or diagonal to lateral ; and a decrease, a change 
from lateral or diagonal to backward. In photonegative colonies, precisely the opposite 
obtains. In photopositive colonies, this results in turning toward and in photonegative 
colonies turning from the source of light. In both, the turning continues until opposite 
sides are equally illuminated, when changes of intensity on the photosensitive tissue are 
no longer produced by rotation and the orienting stimulus ceases. (After Mast, 1926a.) 



302 MOTOR RESPONSES 

pends upon the relative intensity of the Hght in the beams. The higher 
the intensity in one of the beams in relation to that in the other, the 
nearer to the former the point is. If the intensity in the two beams is 
equal, the point is halfway between them. The colonies are oriented 
under these conditions, opposite sides are equally illuminated, both in 
reference to intensity and direction of the rays, i.e., the angle of inci- 
dence at the surface of the colony. If the intensity in the two beams is 
not equal, the illumination of the oriented colonies is higher and the 
angle of incidence greater on one side than on the other. However, when 
a colony is oriented in a field of light, no matter how unequal the in- 
tensity from different directions may be, transfer of the zooids from side 
to side in consequence of rotation on the longitudinal axis causes no 
responses. In other words, the effect of unequal illumination on opposite 
sides is equal. This obviously must be correlated with the difference in 
the angle of incidence. 

Mast (1927a) and Mast and Johnson (1932) demonstrated that the 
location of the point of focus in the eyespot varies with the angle of 
incidence. By ascertaining the location of these points in the eyespots on 
opposite sides of the colonies, in relation to the relative intensity of the 
two beams, they calculated the distribution of sensitivity and found that 
the photosensitive substance is much more sensitive in the central regions 
of the eyespot than at the periphery (Fig. 109). The stimulating effi- 
ciency of light, therefore, depends upon the location of the point of 
focus; this, in turn, depends upon the angle of incidence. The equal effect 
of light on the sides of colonies which are unequally illuminated on op- 
posite sides when they are oriented, is therefore due to the fact that the 
point of focus in the eyespots is more nearly centrally located on the side 
which receives the least light than on that which receives most. 

In photonegative colonies the process of orientation is precisely the 
same as it is in photopositive colonies, except that decrease in intensity 
causes increase in the lateral phase, and increase in the light intensity 
increases the backward phase of the stroke of the flagella. In consequence, 
the illuminated side moves more rapidly than the shaded side. The 
colonies therefore turn away from the light source. 

Orientation of Volvox in light is the result of qualitative differences 
in the action of the locomotor appendages on opposite sides. These dif- 
ferences are due to shock reactions induced by rapid change in the in- 



MOTOR RESPONSES 303 

tensity or the location of the Hght in the photosensitive substance in the 
eyespot, by virtue of colony rotation on the longitudinal axis. It should 
be noted that the responses observed are not the result of quantitative 
differences due to continuous action of the light. The explanation offered 
is therefore not in accord with the Ray-Verworn theory. 

Wave length and response. — The distribution of stimulating efficiency 
in the spectrum for Volvox (Laurens and Hooker, 1920) and Gonium 
(Mast, 1917) is essentially the same as it is for Euglena; but for the 
closely related forms Pandorina and Spondylororum (Mast, 1917) the 
maximum is at 535 ni\\ in place of 485 wp, and the effective region ex- 
tends from this wave length much farther in either direction than it does 
for Euglena, Gomu7n, and Volvox (Fig. 102). The orange, pigmented 
portion of the eyespot in these forms is opaque in reference to the light 
of all those regions of the spectrum which have the highest stimulating 
efficiency. The distribution of stimulating efficiency for these forms con- 
sequently supports the conclusions reached concerning the structure of 
the eyespots, the distribution of photosensitive substance in them, and 
their function in the process of orientation. 

Threshold. — Mast (1907), on the basis of quantitative results, con- 
cludes that the minimum difference in light intensity on opposite sides 
of a colony which is necessary to induce a response varies greatly with 
the physiological state of the colony; but that with colonies in a given 
physiological state, the response varies directly with the intensity, and 
the ratio is nearly constant, i.e.. nearly in accord with the Weber-Fechner 
law. His observations, however, covered such a small range (2-27 meter- 
candles) and the probable error in the results is so large that further 
observations concerning this relation are highly desirable. 

Reversal in response. — Volvox is usually positive in weak, and nega- 
tive in strong light. However, the reverse obtains under some conditions. 
It may be positive, negative, or neutral in every condition of illumination 
in which orientations occurs. If it is positive, a shadow on the photo- 
sensitive substance in the eyespots causes a change in the direction of the 
stroke of the flagella of the zooids from diagonal to backward. A flash of 
light on this substance causes a change of stroke from diagonal to side- 
wise. If the colony is photonegative, the reverse obtains; and if it is 
neutral, there is no response unless the changes in luminous intensity are 
great. 



304 MOTOR RESPONSES 

Reversal in the direction of orientation from positive to negative is 
therefore due to internal changes of such a nature that shock reactions 
which v^'ere produced by decrease are produced by increase in the il- 
lumination of the photosensitive substance. The shift from negative to 
positive is due to the reverse. The nature of the response to light in 
Volvox depends upon the state of adaptation and upon the intensity of 
the illumination. If Volvox is fully adapted in a given intensity, it be- 
comes positive if the intensity is increased or negative if it is decreased. 
If the colony is not fully adapted, it becomes negative if the intensity is 
increased or positive if it is decreased. 

The time required for colonies of Volvox to become negative or posi- 
tive after the luminous intensity has been changed (the reaction time) 
depends upon the degree of adaptation and the extent of the change. 
If colonies which have been subjected first to strong light (1-2 hours) 
and then to a variable period in darkness are exposed to strong light, 
the time required to become positive (reaction time) increases with in- 
crease in the length of the period in darkness (dark adaptation) from 
0.04 minutes (with 2 minutes in darkness) to a maximum of 0.52 min- 
utes (with 16 minutes in darkness) and then decreases to 0.18 minutes 
(with 25 minutes in darkness). If the colonies are kept longer in strong 
light and are then subjected to darkness, the reaction time decreases to 
a minimum and then increases as the time in darkness increases. If they 
are left in darkness until they are fully dark-adapted, and are then ex- 
posed to light of different intensities, the reaction time (as the intensity 
increases) decreases from 29 minutes in 5.24 meter-candles to a mini- 
mum of 0.098 minutes in 7.5 meter-candles, and then increases to 0.358 
minutes in 62,222 meter-candles. The energy required to make the colo- 
nies positive varies directly with the light intensity, over the whole range 
tested. Over most of the range this variation is nearly proportional to the 
variation in intensity. No satisfactory explanation of this relation is avail- 
able. 

If colonies are kept in a given intensity or in darkness, they become 
adapted, i.e., they lose the ability to respond to light. Their responsive- 
ness is regained if, after dark adaptation, the intensity is changed. The 
processes associated with adaptation and those induced by change in il- 
lumination are therefore antagonistic. The rate of these antagonistic 
processes varies greatly, depending upon the magnitude of the change 



MOTOR RESPONSES 305 

in intensity. For example, if dark-adapted colonies are exposed to light 
of 22,400 meter-candles for 0.05 minutes then returned to darkness, it 
takes 20 minutes or more in darkness to eliminate the effect of the light. 
This indicates that, under these conditions, the processes which occur in 
light proceed at least 400 times as fast as the reverse processes which 
occur in darkness. 

To account for the phenomena described, it is necessary to postulate at 
least three interrelated processes, some of which must be directly corre- 
lated with light in such a way that change in illumination of very short 
duration can cause complete reversal in the nature of the response. It is 
altogether probable ( 1 ) that some of these processes are photochemical 
reactions; (2) that others are dependent upon the results of these; and 
(3) that all are closely correlated with the physiological state of the 
organism as^a whole (Mast, 1932b). The evidence now available clearly 
indicates that such simple processes as those postulated by Mast (1907) 
in his first publication dealing with this problem, and those postulated 
by Luntz (1932) are very inadequate (Mast, 1932b). 

A considerable number of other facts have been established concerning 
reversal in Volvox and related forms. For example, increase in tempera- 
ture of hydrogen-ion concentration, and some anesthetics (especially 
chloroform) cause photonegative colonies to become strongly photo- 
positive. However, they usually remain positive only a few moments, 
then become negative again (Mast, 1918, 1919). There is also a very 
interesting correlation between reversal in light and response to elec- 
tricity, in that photopositive colonies always swim toward the cathode 
and photonegative colonies toward the anode (Mast, 1927c). These 
facts show that reversal in light is not due to direct action of environ- 
mental factors. They also indicate that it is correlated with the rate of 
metabolism; but there is no clue to the nature of the processes involved. 

Responses to Electricity 
a. rhizopods 

All the rhizopods which have been investigated {Amoeba, Pelomyxa, 
Difjlugia, Arcella, Actinosphaenmn, and others) respond to electricity. 
Kiihne (1864) and Engelmann (1869) observed that if they are sub- 
jected to a series of induction shocks (alternating current), streaming 
in them stops and they then round up. Verworn (1895), from observa- 



306 MOTOR RESPONSES 

tions on rhizopods in a direct current, maintains that immediately after 
the circuit is closed, there is marked contraction at the anodal side and 
then movement toward the cathode, and that if the current is strong 
enough, disintegration begins on the anodal surface of the organism. 

Greeley (1904), in referring to Amoeba, says that "on the anodal 
side of the cells the protoplasm is coagulated . . . and on the cathode side 
it is liquefied." Bayliss (1920) maintains, however, that the current 
causes only gelation. According to the careful observations of Luce 
( 1926) , hyaline blisters appear on pseudopods oriented with their longi- 
tudinal axis perpendicular to the direction of the current. With the aid 
of superior optical apparatus, he observed the transformation of these 
blisters into pseudopodia. Since there was no indication of gelation, the 
phenomenon must have been due to a liquefaction of the plasmagel at 
the cathodal surface. 

More details concerning response of rhizopods to electricity were ob- 
tained by Mast (1931b) in observations on A. proteus in direct and 
alternating currents of various intensities. The results obtained are as fol- 
lows. 

Direct current. — In direct current of low density, movement continues 
no matter how the amoebae are oriented in the field, but the formation 
of pseudopods is inhibited on the anodal side, resulting in gradual turn- 
ing toward the cathode. In stronger currents movement ceases imme- 
diately after the circuit is closed, then in a few moments one or more 
pseudopods appear on the cathodal side and movement continues directly 
toward the cathode. In still stronger currents there is marked contraction 
on the anodal side immediately after the circuit is closed. This is soon 
followed by disintegration which begins at this side. 

If the anterior end of the amoeba faces the cathode when the current 
is made, there is, in the lowest density that produces an observable effect, 
merely a slight momentary increase in the rate of flow in the plasmasol 
immediately back of the hyaline cap. No change in the rate of flow is 
seen elsewhere. If the current is stronger, this increase extends back 
farther, the hyaline cap disappears, the plasmasol extends to the tip, the 
anterior end becomes distinctly broader, and the plasmagel becomes very 
thin (Fig. 111). If the current is strong enough, this is followed by 
violent contraction at the posterior end, slight contraction at the an- 



MOTOR RESPONSES 



307 



+ 



+ 




+ 





Figure HI. Sketches illustrating the effect of a galvanic current on a monopodal 
Amoeba moving toward the cathode, g, plasmagel; s, plasmasol ; /, plasmalemma ; c, 
hyaline cap; h, hyaline layer; — , cathode; +, anode; arrows, direction of streaming. A, 
very weak current, B, C, D, progressively stronger current. Note that in a current of 
moderate density the hyaline cap disappears and the plasmasol extends to the plasmalemma 
and that in stronger current the cathodal end expands and the anodal end contracts and 
finally breaks, after which the granules in the plasmasol flow toward the anode, indi- 
cating that they are negatively charged. If the surrounding medium is acid, the amoebae 
do not break. (After Mast, 1931a.) 

terior end, and, finally, by disintegration beginning at the posterior end, 
i.e., that which is directed toward the anode. 

If the anterior end of the amoeba is directed toward the anode and 
the current is weak, there is merely a momentary retardation of stream- 
ing at the posterior (cathodal) end. With successively stronger currents, 



308 



MOTOR RESPONSES 



the streaming at this end ( 1 ) stops a few moments, then begins again, 
and continues in the original direction; (2) it stops a bit longer, then 
begins, and continues in the reverse direction, i.e., the plasmasol then 
streams toward the cathode at one end and toward the anode at the other 
(Fig. 112); (3) the reversal extends to the anodal end, and a new hya- 
line cap forms at the original posterior end, which now becomes the an- 
terior end; (4) the reversal is followed by marked contraction at the 



-I- 






-I- 



Figure 112. Sketches illustrating the effect of a galvanic current on monopodal amoebae 
moving toward the anode in a weak current. Labels are the same as in Figure 111. Note 
that the direction of streaming reverses, that it begins at the cathodal end and proceeds 
toward the anodal end, and that this results in movement in opposite directions at the 
two ends during one phase in the process of reversal. (After Mast, 1931a.) 

anodal end, which is followed by partial or complete disintegration, al- 
ways beginning at the anodal end. The fact should be emphasized that 
no matter how extensive a reversal in the direction of streaming may be, 
it always begins at the cathodal end of the amoeba. 

When the contraction at the anodal end begins, the hyaline layer in 
this region becomes thicker here and there, resulting in the formation 
of several small blisters and in numerous minute papilla-like foldings in 
the plasmalemma (Fig. 111). As contraction continues, some of the 
blisters, containing fluid and a few granules in violent Brownian move- 



MOTOR RESPONSES 309 

ment, round up and are pinched off; and others together with the 
plasmagel break, after which granules in the plasmasol stream out and 
proceed rapidly toward the anode. This continues until frequently there 
is nothing left intact except a crumpled membranous sac, the plasma- 
lemma. The plasmagel changes entirely into plasmasol and is carried 
away. The fact that the granules are carried toward the anode shows that 
they are negatively charged. 

McClendon (1910) came to the same conclusion concerning the gran- 
ules in the eggs of frogs and the cells in root tips of onions. But Heil- 
brunn (1923) says: "Particles in the interior of living cells bear a posi- 
tive, whereas the particles in the surface layer have a negative charge." 

If a small amount of HCl is added to the culture solution, the amoeba 
does not disintegrate, regardless of the current strength. Its plasmagel 
turns distinctly yellow at the anodal end immediately after contraction 
begins, after which it increases in thickness until the entire amoeba has 
solidified and is dead. If the current is broken before more than about 
one-fourth of the amoeba has gelated, the gelated portion is usually 
pinched off. The rest of the amoeba then proceeds normally. 

If the amoeba is moving toward the anode when the current is made, 
streaming of the plasmasol reverses before it stops at the anodal end. 
This behavior demonstrates conclusively that the effect of the current 
begins at the surface directed toward the cathode. The fact that before 
the reversal occurs, the thick plasmagel at the cathodal end is replaced 
by a very thin plasmagel sheet and a hyaline cap strongly indicates that 
the first effect of the current is solation of the plasmagel at the cathodal 
surface. This conclusion is supported by the facts that if the anterior end 
is directed toward the cathode when the current is made, the plasmagel 
sheet disappears entirely, the anterior end enlarges, and the plasmasol 
extends to the plasmalemma. It is also true that if the current passes 
through the amoeba in a direction perpendicular to the longitudinal 
axis, the forward streaming stops, and pseudopods are formed on the 
cathodal side. 

The contraction of amoeba at the anodal end, and the increase in the 
thickness of the plasmagel — especially in specimens directed toward the 
anode — seem to show that the current causes gelation at the anodal sur- 
face. 

The facts that the end directed toward the cathode enlarges, that the 



310 



MOTOR RESPONSES 



opposite end decreases in size, breaks up, and becomes yellow just as do 
amoebae killed in an acid solution, and the granules in the amoeba then 
move toward the anode, all indicate ( 1 ) that the granules bear a negative 
charge, ( 2 ) that water in the amoeba is carried toward the cathode, and 
(3) that the plasmagel at the anodal surface becomes acid. 

In very accurately controlled observations on A. proteus, in direct 
current, Hahnert (1932) found that there is at first a momentary in- 
crease in rate of locomotion and then a gradual decrease. He also noted 




Figure 113. Graphs showing comparative effect of different densities of current on the 
rate of locomotion. Abscissae, time in minutes, (A) before and (B) during the passage 
of current; ordinates, apparent rate of locomotion. To obtain actual rate in millimeters 
per minute, divide the apparent rate by 85. (After Hahnert, 1932.) 

that the rate of decrease varies directly with the current density (Fig. 
113), and that the time required to induce cessation of movement in 
specimens directed toward the anode varies nearly inversely with the 
square of the current density, "the time-intensity relation being nearly 
in accord with the equation /^// — (xi) = K in which / is the intensity 
of the current and / its duration." 

Alternating current. — If an active Amoeba mounted in culture fluid is 
subjected to a weak alternating current, locomotion ceases at once. The 
pseudopods are then retracted partially or entirely, and the animal be- 



MOTOR RESPONSES 311 

comes somewhat rounded. It remains in this condition a few moments, 
then a pseudopod appears on one of the sides between the two surfaces 
facing the poles and projects at right angles to a line connecting the two 
poles. Almost immediately after the first pseudopod begins to form, an- 
other usually appears on the opposite surface and extends in the opposite 
direction. Thus the amoeba becomes oriented perpendicularly to the 
direction of the current, i.e., at right angles to the direction in which 
orientation occurs in a direct current. These two pseudopods usually con- 
tinue to stretch out in opposite directions until the amoeba becomes 
greatly elongated. Then one is withdrawn, and the amoeba continues in 
the opposite direction, soon moving out of the field (Fig. 114). 

These pseudopods usually contain no hyaline cap and no plasmagel 
at the tip, and the plasmagel elsewhere is very thin. Sometimes such a 
large portion of the distal end is without plasmagel that the plasmasol 
very definitely streams back at the surface. If the circuit is opened shortly 
after these pseudopods have begun to form, no change in movement is 
seen; if it is closed again, there is still no response. This indicates that 
pseudopods in which the plasmagel is very thin, or absent, do not re- 
spond to electricity; and that the response to electricity consequently is 
due to its action on the plasmagel. 

If the current is stronger, movement ceases and the pseudopods re- 
tract just as they do in weak currents. The contraction which follows 
the retraction of the pseudopods is much more marked, especially on 
the surfaces directed toward the poles. Here the plasmagel fairly shrinks 
up and becomes yellowish in color. This is apparently precisely what 
occurs at the anodal end of an amoeba subjected to the action of a direct 
current. If the circuit is opened immediately after the violent contraction 
has occurred, the amoeba soon recovers. But the two masses of plasma- 
gel that have become yellowish are usually pinched off. An irreversible 
transformation takes place in them, which results in the death of a por- 
tion of the cytoplasm. Occasionally, however, these masses, especially if 
they are relatively small, are taken into the plasmasol and are there di- 
gested. 

If observations are made under an oil immersion objective on a speci- 
men in an alternating current, the plasmagel can be seen to contract and 
become yellowish. Fluid is squeezed out on either side of the organism 
and the adjoining plasmalemma is thrown into folds and papillae of 



312 



MOTOR RESPONSES 



various sizes. The latter are filled with hyaline substance containing a 
few scattered granules which exhibit violent Brownian movement, show- 
ing that the substance in which they are suspended is a fluid with low 
viscosity. Some of the folds and papillae round up and pinch off, to 




Figure 114. A series of camera sketches of an Amoeba, showing the effect of an 
alternating current. A, before current was made; B-F, successive stages after it was 
made; g, plasmagel ; s, plasmasol; /, plasmalemma; h, hyahne layer; c, hyaline cap; 
arrows, direction of streaming; double headed arrows, direction of the current. Note 
that the Amoeba orients perpendicularly to the direction of the current, that the plasmagel 
in the pseudopods is at this time very thin or absent, that the plasmagel contracts violently 
at the surface directed toward the poles, that blisters are formed on these surfaces, and 
that the Amoeba eventually breaks here and then disintegrates. If the surrounding medium 
is acid, the Amoeba does not break and disintegrate. (After Mast, 1931a.) 

form spherical bodies filled with granule-containing fluid observed in 
the folds (Fig. 114). 

If the current is strong enough, the plasmagel usually breaks after 
it has thus contracted. The plasmasol flows out and the entire amoeba 
soon disintegrates and dissolves. Sometimes breaks occur in the pseudo- 



MOTOR RESPONSES 313 

pods before there is much contraction, and then the plasmasol flows out 
through these. As the plasmasol flows out, it collects about the amoeba; 
and the granules and fluid in it do not stream toward the poles as they 
do in direct current. Cataphoresis and electroendosmosis are thus neutral- 
ized, owing to the reversal in the direction of flow of the current. 

There is no change in the responses if acid is added to the culture fluid. 
However, instead of disintegrating as usual, the plasmasol coagulates 
after the amoeba breaks up. 

The essential phenomena observed in the effect of the alternating 
current on Amoeba appear to be: (1) mild contraction in extended 
pseudopods, beginning at the tip; (2) violent contraction on the two 
surfaces facing the poles, with the formation of blisters in these regions; 
(3) formation of highly fluid pseudopods between these two surfaces; 
and, finally, (4) rupture at the surfaces directed toward the poles, fol- 
lowed by disintegration of the organism. 

Contraction in the pseudopods, as was repeatedly observed, occurs 
first in those directed toward the poles and last in those in which the 
longitudinal axis is perpendicular to the direction of the current. In- 
deed, there is some indication that there is no contraction at all if the 
axis is actually perpendicular to the direction of the current. Contraction 
in the pseudopods under these conditions results in retraction which, 
in all respects, appears to be the same as retraction of pseudopods in 
normal locomotion. The retraction appears to be due to increase in the 
elastic strength of the plasmagel, owing to reversible gelation of ad- 
joining plasmasol. It may be concluded, then, that the first effect of the 
alternating current is reversible gelation of the plasmasol adjoining the 
plasmagel at the tip of the pseudopods directed toward the poles. This 
results in an increase in elastic strength of the plasmagel in this region, 
and in a retraction of the pseudopods. The contraction at the surfaces 
facing the poles is, in the beginning, doubtless due to the same phe- 
nomena; but the facts ( 1 ) that the plasmagel in this region later changes 
in color, ( 2 ) that it does not become thicker, ( 3 ) that fluid is squeezed 
out of it, and (4) that it is killed and then breaks, show that contraction 
here is associated with profound changes in the plasmagel itself. These 
changes result in such marked decrease in the strength and elasticity of 
this structure that it breaks readily. These changes are also associated 
with simultaneous increase in fluidity of the plasmasol, as indicated by 



314 MOTOR RESPONSES 

the structure of the pseudopods which form at this time. The evidence 
presented above shows that the described changes are brought about by 
the action of the current. 

Alsup (1939) measured the time required in alternating current and 
in Hght respectively to cause cessation in streaming (reaction time) and 
that required for recovery after this response (recovery period). He 
found in both that the reaction increased as the recovery period decreased. 
This indicates that after the plasmasol has gelated, owing to the action 
of electricity or light, and then solated, owing to the recovery processes, 
it no longer gelates so readily as it did. 

Alsup also found that subminimum exposure to an alternating cur- 
rent followed by a subminimum exposure to light, or vice versa, may 
induce a response, indicating that the effects of these two agents are 
additive. 

In order to account for the essential phenomena observed in amoebae 
when subjected to the action of an alternating current, it is then neces- 
sary to explain reversible gelation of the plasmasol adjoining the plasma- 
gel on the sides of the organisms facing the poles. It should be noted 
that the gelation of the plasmasol is followed by changes in the plasmagel 
in the adjoining regions — changes which result in violent contraction, 
loss of fluid, decrease in elasticity, and rupture, and by increase in the 
fluidity of the plasmasol. 

Mechanics of response. — In rhizopods all of the responses to direct 
current appear to be due primarily to solation at the cathodal surface, 
followed by gelation at the anodal surface. The question then arises as 
to what causes this. 

If an electric current is passed through a culture solution containing 
amoebae, the negative ions in the solution and in the amoebae move to- 
ward the anode, whereas the positive ions migrate toward the cathode. 
If the surface layers of the amoebae are semipermeable, as they un- 
doubtedly are, there will be an accumulation of positive ions (e.g., Na) 
on the inside, and negative ions (e.g., CI) on the outside of the surface 
of the amoebae directed toward the cathode. Positive ions will accumulate 
on the outside and negative ions on the inside of the surface directed 
toward the anode (Ostwald, 1890). The positive ions will, however, 
unite with the hydroxyl ions of the water, forming bases (e.g., NaOH) ; 
and the negative ions will unite with the hydrogen ions of the water, 



MOTOR RESPONSES 315 

forming acids (e.g., HCl). The cathodal surface layer of the amoebae 
should therefore become alkaline on the inside and acid on the outside. 
The anodal surface layer should react in the opposite manner. 

Numerous observations were made on specimens stained with neutral 
red and subjected to direct current under various conditions. No differ- 
ence whatever was observed in the color of different regions in any of 
these specimens. The neutral red staining in A. proteus is, however, 
confined to granules and vacuoles. It was observed that if the specimens 
are crushed, the color of these granules and vacuoles does not immedi- 
ately change, in accord with the hydrogen-ion concentration of the solu- 
tion in which they are immersed. It is obvious, then, that the fact that 
no difference in color was observed in the vacuoles and granules does not 
prove that the hydrogen-ion concentration of the cytoplasm was the 
same. Moreover, there is indirect evidence which indicates that it was 
not the same. 

Kiihne (1864) long ago observed in certain epidermal cells of the 
leaves of Tradescantia subjected to a galvanic current that the ends of 
the cells directed toward the cathode become alkaline, and that those 
directed toward the anode become acid. These cells contain a natural 
indicator which is bluish in neutral solutions, red in acid solutions, and 
green in alkaline solutions. 

Habenicht (1935) came to the same conclusion in experiments on the 
effect of the galvanic current on cylinders of egg white. 

Mast (1931b) repeated and extended Kiihne's experiments and ob- 
tained results which confirm his contentions. It may then be assumed with 
considerable confidence that when an amoeba is subjected to a direct 
current, the hydrogen-ion concentration in the cytoplasm decreases at 
the cathodal end and increases at the anodal end. 

Edwards (1923) demonstrated that if an alkaline solution is locally 
applied to the surface of an amoeba, the plasmagel in this region dis- 
integrates; and that if acid is applied, it becomes thicker, owing to gela- 
tion of the adjoining plasmasol. This has been confirmed indirectly by 
Pantin (1923), Chambers and Reznikoff (1926), and others. If, then, 
the direct current produces a decrease in hydrogen-ion concentration at 
the cathodal end and an increase at the anodal end, one would expect 
the plasmagel to become thinner at the cathodal end and thicker at the 
anodal end. This is precisely what was observed. And if the elastic 



316 MOTOR RESPONSES 

strength of the plasmagel varies directly with its thickness, as is doubt- 
less true, this would result in formation of pseudopods at the cathodal 
surface. This has been confirmed by observation. Since streaming toward 
the cathode begins at the cathodal surface before it does at the anodal 
surface, movement toward the cathode must be due primarily to the 
solation of the plasmagel at the cathodal surface. The accumulation of 
positive ions at this surface therefore must produce the solation. But this 
obviously does not account for the disintegration of the entire organism. 
Neither does it account for the violent contraction preceding disintegra- 
tion at the anodal side. It will be remembered that violent contraction 
and disintegration beginning at the anodal surface were observed only 
in relatively strong currents. Furthermore, the reactions were observed 
to occur only after large pseudopods develop and begin to advance to- 
ward the cathode. 

An amoeba disintegrates only if the direct current applied is suffi- 
ciently strong. After the circuit is closed, there is, on the inner surface of 
the plasmagel or in the plasmagel, an accumulation of positive ions at 
the cathodal side, and of negative ions at the anodal side. The former 
produces a decrease in the elastic strength of this layer, which results in 
the formation of a pseudopod directed toward the cathode — a pseudopod 
in which the plasmagel extends to the plasmalemma. Local disintegra- 
tion occurs at first; but, as the current continues, more and more of the 
plasmagel in this pseudopod disintegrates. The accumulation of nega- 
tive ions and consequently of hydrogen ions at the anodal end causes a 
thickening of the plasmagel, as well as gelation of the adjoining plas- 
masol. This results in violent contraction and finally in the rupture ob- 
served at the anodal end. 

Cataphoresis and electroendosmosis are probably also involved. The 
granules in Amoeba are negatively charged in relation to the fluid. The 
fluid consequently tends to flow from the anodal toward the cathodal 
end. This would facilitate contraction at the former and expansion at the 
latter end, which is precisely what was observed. There is, however, some 
evidence to indicate that transfer of water is of little importance in the 
rupture and disintegration at the anodal end. This will be presented later. 

It is therefore fairly clear how, in a direct current, substances accumu- 
late locally; and how this can produce most of the processes associated 
with the responses of amoebae in it. But in an alternating current the 



MOTOR RESPONSES 317 

situation is quite different. There is an equal movement of all substances 
in opposite directions. Consequently there can be no accumulation of 
different substances in different parts of the organism, unless there is 
some process which makes the movement in one direction greater than 
that in the other. 

The essential phenomena observed in Amoeba as a consequence of 
exposure to alternating current may be summarized in the order of their 
appearance. A mild contraction begins first at the tips of extended pseudo- 
podia. This is followed by violent contraction on the two surfaces fac- 
ing the poles (blisters appear on these surfaces). Then highly fluid 
pseudopods form between these two surfaces. Finally, the surfaces di- 
rected toward the poles rupture and the organism disintegrates. 

Numerous observations with the best lens system obtainable were 
made on the movement of microscopic particles, both in the field of the 
alternating current and in the amoebae in this field. There was no indi- 
cation of a drift of these particles nor of their accumulation in any part of 
the organisms. It therefore is evident that cataphoresis and electroendos- 
mosis cannot be involved in the observed contraction. It seems necessary, 
then, to conclude that the phenomenon of contraction is associated with 
the movement of the ions produced by the electric current; and, further, 
that ion movements are accompanied by processes which result in the 
accumulation of ions in certain regions of the organism. 

Dixon and Bennet-Clark (1927) and others maintain that alternat- 
ing current causes increase in the permeability of the plasmamembrane 
in cells. If so, then may not the contraction observed in the plasmagel be 
due to the action of substances which enter from the surrounding medium, 
since localized accumulation of ions at the surface of the amoeba in- 
creases its permeability owing to the action of the current? 

Two facts suggest that the contraction of Amoeba during exposure to 
alternating current cannot be due to the entrance of substances from the 
outside. In the first place, the contractions are known to occur in both 
alkaline and acid solutions. Secondly, alkaline solutions tend to produce 
solation in the plasmagel, thus decreasing its elastic strength. Possibly 
the accumulation of ions in or near the plasmagel causes the contrac- 
tion. 

It is well known that the positive ions, Na, K, Ca, and others ordi- 
narily pass through membranes more readily and more rapidly than the 



318 MOTOR RESPONSES 

negative ions, SO^, PO^, NO3, and others. In a structure like the plas- 
magel, the movement of ions is undoubtedly hindered. Nevertheless, 
the positive ions may move farther from their initial positions toward 
the pole than do the negative ions. It may be assumed that the nega- 
tive ions tend to remain in the plasmagel, whereas the positive ions 
tend to leave it and return again as the direction of the current reverses. 
If the return movement of the positive ions is inhibited, there may be 
a momentary preponderance of negative ions within the plasmagel, 
and of positive ions in the adjoining substances, i.e., in the plasmasol, 
the hyaline layer, and the plasmalemma. If the movement of the posi- 
tive ions away from the negative ions is extensive enough, the negative 
ions remaining in the plasmagel will unite with the hydrogen ions of the 
water surrounding them, to form acids. The hydroxyl ions thus lib- 
erated will, owing to the fact that they pass rapidly and freely through 
tissues, move out and unite with the positive ions, to form bases in the 
substance adjoining the plasmagel. It is possible that this union retards 
the return movement of the positive ions during the next reversal in the 
direction of the current. Consequently the effect would be cumulative, 
gradually increasing the acidity within the plasmagel and the alkalinity 
of the substance on either side. 

The increase in acidity in the plasmagel would produce gelation in this 
layer and probably also in closely applied plasmasol. This would in- 
crease the thickness and elastic strength of the plasmagel. Contraction 
would be the result. If the accumulation of negative ions were great 
enough, the increase in acidity in the plasmagel would cause irreversible 
coagulation (death), accompanied by violent contraction and dehydra- 
tion, thus making the coagulated plasmagel so brittle that it would break 
readily. The increased alkalinity in the plasmasol and plasmalemma 
would tend to make the former more fluid, and it would tend to break 
up the latter. The postulated action of the current is precisely in accord 
with the observations. It also accounts for the fact that no response was 
observed in pseudopods which contained no plasmagel. According to the 
explanation offered, an accumulation of ions occurs in or near the plas- 
magel; there could be no action in structures which have none. 

Moreover, on the basis of this hypothesis, it is possible to account 
for the well-known fact that in many organisms the effect of a current 
varies inversely with the frequency of reversal. It is necessary only to as- 



MOTOR RESPONSES 319 

sume that the higher the frequency of reversal, the more restricted the 
movements of the ions toward the poles, and the shorter the period of 
separation of positive from negative ions; and that the shorter this period, 
the more restricted the union of the negative ions with hydrogen ions, 
and the positive ions with hydroxyl ions. The more restricted these unions, 
the less the increase in acidity in the plasmagel, and the less the increase 
in alkalinity in the plasmasol, and the less the stimulating and the in- 
jurious effect. The hypothesis, then, that the action of the electric cur- 
rent on organisms is due to localized increase in acidity and alkalinity 
in different regions of the cell is in full accord with the fact that the 
effect in alternating currents varies inversely with the frequency. 

What bearing has all this on the problem concerning the observed con- 
traction at the anodal side of amoebae subjected to direct current? 

Carlgren (1899), as previously stated, holds that this is due to elec- 
troendosmotic extraction of water by the current, owing to negative 
charge of the solid substance. However, the fact that the same phe- 
nomenon occurs in alternating current, in which electroendosmosis is neu- 
tralized, strongly indicates that Carlgren's conclusion is not valid. It also 
seems to show that the anodal contraction in direct current must be due, 
as appears to be the case in alternating current, to the action of the cur- 
rent on the movement of ions. 

The evidence presented in reference to the effect of both direct and 
alternating current on A. proteus indicates that the assertion of Bayliss 
(1920), Weber and Weber (1922), Taylor (1925), and others that 
electricity gelates cytoplasm, is misleading, for it shows that if an electric 
current causes gelation in a cell, it probably always causes simultaneous 
solation, each being confined to a portion of the cell. 

It was demonstrated above that this does not obtain for light. The 
implication frequently found in the literature that the action of electricity 
on protoplasm is the same as the action of light, appears therefore to be 
erroneous. 

Heilbrunn and Daugherty ( 1931 ) found that if ammonium hydroxide 
or chloride is added to the culture fluid, A. proteus becomes anopositive. 
They maintain that the "protoplasmic granules" are ordinarily positively 
charged and are consequently carried (cataphoretically) toward the 
cathode, and that ammonium hydrate or chloride causes a change in 
the charge to negative and a consequent reversal in the direction in which 



320 MOTOR RESPONSES 

they are carried. They contend that the contact of these granules with 
the inner surface of the plasmagel causes it to hquefy, and that this 
results in the formation of a pseudopod which is directed toward the 
cathode if the granules are positively charged and toward the anode if 
they are negatively charged. These authors say: 

As the granules move either toward cathode or anode, they must tend to 
break down the thixotropic gel on the side toward which they move. . . . 
If this gel is liquefied in any local region, such a region becomes pushed 
out to form an advancing pseudopod. 

These are interesting views, but they obviously do not account for 
the direction of movement of pseudopods in alternating current (Fig. 
114), nor for the direction of movement of pseudopods under some 
conditions in direct current (Fig. 112). For this would require cata- 
phoretic movement of the granules perpendicularly to the direction of 
the current in the former, and in opposite directions at opposite ends 
of the amoeba in the latter. Moreover, the fact that the granules stream 
toward the anode after the amoeba disintegrates, indicates that they are 
normally negatively (not positively) charged, and that this charge is 
consequently not involved in the cathopositive response. How, then, can 
the observed reversal in the direction of orientation be explained? 

The cathopositive response is probably brought about as described 
above. The ammonium hydrate or chloride added to the culture fluid 
probably results in the liquefaction of all the plasmagel, and conse- 
quently in free movement toward the anode of the negatively charged 
granules suspended in it. According to this view, the reversal in the 
direction of galvanic orientation is due to liquefaction of the plasmagel 
caused by the ammonium compounds used, not to change in the electric 
charge on the "protoplasmic granules." 

B. FLAGELLATES 

Some of the flagellates orient very precisely in a direct current. Some 
are cathopositive, others anopositive, and still others both or neutral, 
depending upon the environmental conditions (Verworn, 1889; Pearl, 
1900; Bancroft, 1913). Verworn maintains that orientation is brought 
about by differences in the effective stroke of the flagellum in opposite 
directions. Pearl believes that it is the result of typical avoiding reac- 



MOTOR RESPONSES 321 

tions, whereas Bancroft thinks that it is identical with the process of 
orientation in hght. Further details concerning the processes involved 
are much desired. Moreover, it is noteworthy that some of the flagel- 
lates have proved to be excellent material for quantitative study of the 
relation between stimulus and response. 

C. CILIATES 

The responses to electricity have been more intensively and extensively 
studied in the ciliates than in any of the other groups of Protozoa. 

Jennings (1906) presents an excellent review of all the earlier in- 
vestigations concerning these responses. He maintains that the results 
obtained show the following: 

The principal feature in the response of all of the different species 
studied consists of reversal in the direction of the effective stroke of the 
cilia on the cathodal surface. In those species in which other ciliary 
actions are only slightly or not at all involved (e.g., Paramecium) , this 
results in direct turning until the anterior end is directed toward the 
cathode. The organism then moves toward this pole. The extent of the 
cathodal surface affected varies directly with the strength of the current. 
If the current is strong enough to produce reversal over more than half 
of the surface of the Favamecium, it swims backward toward the anode. 
A still stronger current causes marked swelling of the anterior end and 
contraction of the posterior end, changes which are followed by disin- 
tegration beginning posteriorly (Fig. 115). The effect of the reversal 
on the cathodal surface is variously modified by the normal action of 
the cilia in other regions, in such a way that "with different strengths of 
current, and with infusoria of different action systems, this results some- 
times in movement forward to the cathode; sometimes in movement 
forward to the anode; sometimes in cessation of movement, the anterior 
end continuing to point to the cathode; sometimes in a backward move- 
ment to the anode; sometimes in a position transverse to the current, the 
animal either remaining at rest or moving across the current." 

Jennings holds that all the responses of the ciliates to electricity are 
due to stimulation at the cathodal surface, resulting in local reversal in 
the direction of the effective stroke of the cilia on this surface. 

Bancroft (iyo6), in his experiments with Paramecium, observed that 
if certain salts (especially potassium, sodium, or barium salts) are added 



322 



MOTOR RESPONSES 



to the solution, the organisms will swim forward toward the anode. He 
maintains that this is due to stimulation at the anodal surface, and that 
consequently it is an exception to Pfliiger's law. 

It is well known that some salts cause paramecia to swim backward, 




Figure 115. Progressive cathodic reversal of the cilia and change of form in Para- 
inecium as the constant electric current is made stronger. The cathode is supposed to lie 
at the upper end. The current is weakest at 1, where only a few cilia are reversed; 2-6, 
successive changes as the current is gradually increased. (After Statkewitsch, 903.) 



owing to the forward stroke of all the cilia (Jennings, 1899; Mast and 
Nadler, 1926; Oliphant, 1938). Obviously, if under such conditions 
there is reversal in the direction of the stroke at the cathodal surface, 
the paramecia will turn and swim forward toward the anode (Mast, 



MOTOR RESPONSES 323 

1927c). If this reaction obtains, the forward swimming toward the 
anode is in full accord with Pfliiger's law. 

Kamada (1929) made a study of the correlation between the effect 
of many different salts on reversal from forward to backward swimming, 
and the direction of orientation in a direct current. He maintains that 
some salts which induce forward swimming toward the anode do not 
induce backward swimming. He consequently supports Bancroft's views. 

Kamada (1931) also maintains that in paramecia which are ano- 
positive there is ciliary reversal at the anodal surface, in place of the 
cathodal, and that with increase in current density this is modified in 
various ways. The evidence he presents is, however, by no means con- 
clusive. Further observations are therefore needed. 

Paramecia are most sensitive if the anterior end is directed toward the 
cathode, less sensitive if it is directed toward the anode, and least sensi- 
tive if the longitudinal axis is perpendicular to a line connecting the two 
poles ( Statkewitsch, 1903; Kinosita, 1936). The same holds for Spi- 
Yostomum (Kinosita, 1938a). 

Statkewitsch (1907) subjected to direct and to alternating currents 
paramecia which had been stained with neutral red. He maintains that the 
stained structures in them became violet (acid) in weak currents and 
distinctly yellowish (alkaline) in strong currents. He apparently did not 
observe any difference in the color at the two ends. In Kinosita's (1936) 
experiments with paramecia stained with either neutral red or Nile blue 
sulphate, the color of opposite ends differed. He says that the changes 
in color observed show that the paramecia become acid at the cathodal 
and alkaline at the anodal end, but that the alkaline portion rapidly ex- 
tends forward and soon includes the entire body. 

There is consequently a diversity of opinion concerning the effect of 
the electric current on the hydrogen-ion concentration of the cytoplasm in 
Paramecium. This also obtains for other cells, since, as previously stated, 
Kiihne maintains that cells of Tradescantia become acid at the anodal end 
and alkaline at the cathodal end. Mast confirmed this, but could observe 
no difference in this respect between the two ends in Amoeba. It is there- 
fore obviously desirable to have further observations concerning this 
problem, for it is theoretically very important. 

Kinosita measured the time required to make the anodal end alkaline 



324 MOTOR RESPONSES 

in different current densities, extending over a wide range. He maintains 
that the results indicate that i\/t — a) = K. This equation is similar 
to the one obtained by Kamada in observations on the destruction of the 
surface membrane in paramecia by a direct current. It is also related to 
the one obtained by Hahnert (see above) in his observations on the 
cessation of streaming in Amoeba in a constant current. 

Internal processes involved in response. — Several theories have been 
formulated to account for the responses of ciliates to an electric current. 
Loeb and Budgett (1897) contend that these are in reality the result of 
responses to changes in the chemical constitution of the environment 
produced by the electric current. Pearl (1901) asserts that the direction 
of the stroke of the cilia is specifically correlated with the direction of 
protoplasmic streaming directly below the surface. Coehn and Barratt 

(1905) hold that the movements are purely cataphoretic; Bancroft 

(1906) maintains that the galvanic responses are due to local changes 
in the calcium content of the tissue in relation to that of other ions, 
especially monovalent cations. Although Carlgren (1899) lays especial 
stress on localized changes in water content within the organisms, due 
to endosmotic streaming. Some assert that the movements are due to 
direct action of the electric current on the cilia, others that they must 
be due to action on a coordinating center. Ludloff (1895), Verworn 
(1895 ) , and Koehler ( 1925 ) postulate functional division of the organ- 
isms into anterior and posterior halves, such that one responds in one 
way that the other responds in another way. The views of Nernst ( 1899) , 
Lucas (1910), Lillie (1923), and others regarding stimulation in higher 
organisms would lead to the idea that local changes in permeability of 
the surface membrane is the all-essential in controlling the movements 
of the lower organisms in an electric field. 

The relation between these hypotheses and the facts established has 
been very illuminatingly discussed by Jennings (I906) and Koehler 
(1925). Both conclude that the facts in hand are not adequately ac- 
counted for by any of the hypotheses presented. Jennings contends, as 
previously stated, that the most important perceptible characteristic of 
the response is reversal in the direction of the stroke of the cilia on the 
cathodal side. Koehler holds that the processes involved cannot be solely 
dependent upon surface phenomena, that somehow the current results 
in a division of the organism into cathodal and anodal portions which 



MOTOR RESPONSES 325 

function differently, owing to different internal factors. But neither Jen- 
nings nor Koehler offers any explanation of how the responses are reg- 
ulated. 

Among the most important of the known facts concerning the re- 
sponses in Protozoa to an electric current are those discovered by Lud- 
loff in observations on ParamecJutn. Ludloff (1895) found, as has been 
abundantly confirmed, that when the circuit is closed, the direction of 
the stroke of the cilia on the surface of the paramecia directed toward 
the cathode reverses; but that if the longitudinal axis of the organisms 
is directed obliquely to the direction of the current, reversal occurs on 





Figure 116. Paramecium showing reversal in the direction of the stroke of the cilia 
in a galvanic current. A, weak current; B, strong current; +, anode, — , cathode. (After 
Ludloff, 1895.) 

all sides of the end of the body nearest the cathode, extending to a line 
around the body produced by passing a plane through it at right angles 
to the direction of the current. The extent of the portion of the body 
on which such reversal occurs depends upon the strength of the current: 
the stronger the current, the larger the portion affected (Fig. 116). 

The fact that the cilia in different regions on the same side of the 
paramecia are not always affected equally by the current seems to show 
that the responses observed cannot, as Koehler points out, be due to 
direct action on the cilia or to surface phenomena alone; for if they were, 
all the cilia on either side should act alike, with the possible exception 



326 MOTOR RESPONSES 

of those in the oral groove. If this is true, it is evident that the responses 
in this form must be associated with internal changes. 

The fact that in specimens with the longitudinal axis directed ob- 
liquely to the direction of the current, the entire end nearest the cathode 
is affected, and the fact that the size of the portion affected varies directly 
with the strength of the current, indicates not only that the current re- 
sults in a functional division of the organism (as maintained by Ludloff, 
Verworn, and Koehler), but also that whatever the factors involved 
may be, they act on a structure which is well distributed through the 
entire body and which is located some distance below the surface — 
probably the neuromotor apparatus. For only in a structure which is 
some distance from the surface, could a current produce the same changes 
in the distribution of substances on the anodal and the cathodal sides 
of the portion affected, resulting in reversal in the direction of the 
stroke in all the cilia on this portion. 

It is well known that momentary reversal in the stroke of the cilia 
can be induced in Paramecium by almost any sudden environmental 
change (Jennings, 1906), and that more prolonged reversal can be in- 
duced by transfer from culture fluid to distilled water or from distilled 
water to solutions of monovalent cation salts, but not usually by transfer 
to solutions of bivalent cation salts (Mast and Nadler, 1926; Oliphant, 
1938) . These changes, therefore, produce the same result as is produced 
by an electric current on the cathodal surface, indicating similarity in 
action. 

Greeley (1904) maintains that paramecia drift toward the anode, i.e., 
that they are negatively charged, indicating that there is a negative layer 
at the surface. Statkewitsch (1903) observed that if one end is directed 
toward the anode and the other toward the cathode, the former shrinks 
and the latter swells, indicating that the more solid substance is negative 
in relation to the more fluid substance. If all this obtains, a direct cur- 
rent will result in a decrease in the concentration of the positive, or an 
increase in the concentration of the negative ions on the cathodal side of 
the surface of the organism and on each semipermeable structure within. 
There will also be a decrease in the concentration of the negative, or an 
increase in the concentration of the positive ions on the opposite side. As 
a result, water will drift toward the cathodal side, and the solid particles 
will drift in the opposite direction. 



MOTOR RESPONSES 327 

Since reversal in the stroke of the ciHa begins on the cathodal side, 
it would seem that it must be associated either with an increase in the 
concentration of the negative ions or with a decrease in the concentration 
of the positive ions, on the cathodal side of the semipermeable structures 
on this side of the organisms. Either a decrease of polarization or an 
increase in the water content of the cytoplasm may be involved. If this 
is true, then transfer from culture fluid to distilled water and from dis- 
tilled water to solutions of monovalent cation salts should, since this re- 
sults in reversal of the stroke of all the cilia, produce similar changes 
in the concentration of ions or water. In other words, the transfer from 
culture fluid to distilled water should produce a decrease in the con- 
centration of positive ions on the outside of the semipermeable struc- 
tures, or an increase in the water content at the surface. A transfer from 
distilled water to solutions of monovalent cation salts should produce 
like changes. Whether or not this obtains is at present unknown, but one 
would expect it to obtain, if, as is frequently asserted, permeability is 
increased by monovalent cation salts. 

D. COLONIAL ORGANISMS 

No detailed observations have been made on the response to electricity 
of any of the colonial organisms except Volvox. Volvox orients very 
precisely in direct current. It swims toward the cathode under some condi- 
tions and toward the anode under others. Carlgren (1899) maintains 
that reversal in the direction of orientation is correlated with the dura- 
tion of exposure to the current. Terry (1906) and Bancroft (1907) 
contend that it is correlated with the intensity of the light received and 
the duration of exposure to it. Mast (1927c) found that Volvox swims 
toward the cathode when it is photopositive, and toward the anode when 
it is photonegative, i.e., that the response to electricity is specifically cor- 
related with the response to light. 

In photopositive colonies in which rotation on the longitudinal axis 
is inhibited by means of pressure, the flagella on the cathodal side stop 
beating immediately after the circuit is closed. They remain inactive 4.5 
to 6 seconds and then begin to beat again (Fig. 117). If the circuit 
is now opened, those on the anodal side stop for a few moments and 
then beat again. If the colonies are swimming and rotating on the longi- 
tudinal axis in the normal way, but are not proceeding directly toward 



328 



MOTOR RESPONSES 



either pole when the current is made, flagellar inactivity on the cathodal 
side is continuous, owing to the continuous transfer of the zooids from 
the anodal to the cathodal side. The colonies therefore turn toward the 
cathode until they face it directly, and the transfer of zo5ids from side 
to side ceases. 

Orientation. — In photonegative colonies precisely the opposite occurs. 
The flagella on the anodal side stop beating after the circuit is closed. 




+ 




Figure 117. Sketch showing in a stationary photopositive colony of Volvox the effect 
of a galvanic current on the currents of water produced by the flagella. A, outline of 
colony oriented in light; B, same colony immediately after the circuit was closed; a, 
anterior end; straight arrows, direction of illumination; curved arrows, currents pro- 
duced by the flagella; +. positive pole; — , negative pole. (After Mast, 1927.) 

and the colonies turn toward the anode until they face it and then swim 
toward it (Mast, 1927c). 

Galvanic orientation is consequently correlated with photic orientation, 
but the processes involved diff^er, for, as previously stated, photic orienta- 
tion is due to a change in the direction of the stroke of the flagella on 
opposite sides, while galvanic orientation is due to decrease or cessation 
in the activity of the flagella on one side. 

Electric charge on the colonies. — Galvanic orientation in Volvox also 
differs from that in Farajuecium, for while the one is due to decrease or 
cessation in activity on one side, the other, as previously stated, is due to 
reversal in the direction of the eff^ective stroke of the cilia on one side. 

Since a given colony of Volvox may be either photopositive or photo- 
negative in the same environment, the difference in response to the light 



MOTOR RESPONSES 329 

in this environment must be due to changes in the colony itself. Since 
photopositive response is specifically correlated with cathopositive re- 
sponse, and photonegative response with anopositive response, the differ- 
ence in the response to electricity must be due to like changes in the 
colony. The only difference observed in the colonies in connection with 
the response to light concerns the electric charge. Referring to this, Mast 
(1927c) says: 

Most of the photopositive colonies observed drifted toward the anode 
and most of the photonegative ones drifted toward the cathode, indicating 
that the former were negatively and the latter positively charged. However, 
owing to the negatively charged glass bottom of the aquarium in which 
the observations were made, there was produced in the solution near the 
bottom an endosmotic current of water toward the cathode and this current 
produced at the upper surface a current in the opposite direction, i.e., toward 
the anode. In making the observations it was impossible to ascertain pre- 
cisely the location of the colonies in relation to these currents, resulting fre- 
quently in uncertainty as to whether the drift was due to cataphoresis or 
to endosmosis. The results obtained are consequently somewhat equivocal. 

These observation should therefore be repeated under more favorable 
conditions, for the results are of fundamental importance in the analysis 
of the mechanics of the response to electricity, as will be shown pres- 
ently. 

Mechanics of response. — The outstanding characteristics of the re- 
sponses to the electric current of the colonial forms, exemplified in 
Volvox, consist in momentary decrease in the action of the flagella, cor- 
related with the direction and the density of the current and the nature 
of the response to light. In photopositive colonies this occurs in such a 
way that the flagellar activity decreases on the cathodal side after the 
current is made. In photonegative colonies the activity decreases on the 
anodal side, continues a few seconds, and then increases again. But if 
the current is broken, it decreases on the opposite side, continues a few 
seconds, then begins again. The extent of the region affected under all 
conditions varies directly with the density of the current. 

The action of the current must be due to movement of ions, parti- 
cles, or fluid in the colonies or the surrounding solution, and to differ- 
ences in the responses in photonegative and photopositive colonies to 
differences in the effects produced by the movements of these substances. 



330 



MOTOR RESPONSES 



There is considerable evidence which indicates that when colonies are 
photopositive and cathopositive, they are negatively charged; and when 
they are photonegative and anopositive they are positively charged 
(Mast, 1927c). If this obtains, the movement of ions in photopositive 
colonies in a direct current would result in decrease in the negative ions 







K 









K 



K 




K 




A 




Figure 118. Diagrams illustrating the effect of direct current on the distribution of 
ions in colonies of Volvox and their response. — , negative charge on colony; +, positive; 
o, no charge; A, anode; K, cathode; large arrows, beam of light; small arrows, direction 
of movement of colonies in the beam of light; curved arrows, direction the colonies turn 
after the current is made and broken respectively. Note that there is in negatively charged 
colonies, after the circuit is closed, a decrease in potential at the cathode, and a decrease 
at the anode after it is opened, that the opposite obtains for positively charged colonies, 
and that there is no change in potential in neutral colonies. (Modified after Mast, 1927b.) 

on the cathodal side. In turn, this would cause decrease in potential and 
increase in permeability here. It would also result in increase in the 
negative ions at the anodal side and increase in potential and a decrease 
in permeability there (Fig. 118). The movement of ions in photonega- 
tive colonies would result in increase in positive ions, in the potential, 
and increase in permeability on the cathodal side. The reverse would 



MOTOR RESPONSES 331 

occur on the anodal side. The ion movement in photoneutral colonies 
would result in increase in positive ions, and increase in potential and 
decrease in permeability on the cathodal side. On the anodal side there 
would be equivalent increase in negative ions and increase in potential 
and decrease in permeability. After the current is broken, the change 
in distribution of ions, and its effect, would be precisely opposite in all 
respects (Fig. 99). 

If the decrease in flagellar activity is due to local decrease in polariza- 
tion and increase in permeability, it accounts for the observed direction 
of movement of photopositive and photonegative colonies in a direct 
current. And if the decrease in flagellar activity is correlated with rate 
of change in these characteristics, it also accounts. for the fact that the 
decreased activity induced by making or breaking the current continues 
only a few seconds, for the change in polarization and permeability 
undoubtedly lasts but a few seconds. 

How does the fact that strong currents cause decrease in the activity 
of the flagella simultaneously on all sides of the colonies, harmonize with 
this view? 

It is well known that a galvanic current will produce cytolysis if it is 
strong enough, and that cytolysis is associated with increase in perme- 
ability and decrease in polarization. It is therefore not difficult to see 
that such a current could cause decrease in polarization simultaneously 
on all sides. 

It is probable, however, that the processes involved in galvanic stimula- 
tion, resulting in orientation, are not the same in all organisms. For 
example, Ludloff (1895) and Statkewitsch (1903) found that in a 
galvanic current the fluid in the body of Paramecium is carried endosmoti- 
cally toward the surface, on which the stroke of the cilia reverses, i.e., 
in the specimens which swim forward toward the cathode, there is, if 
the current is strong enough, contraction at the anode and expansion 
at the cathode end. In Volvox precisely the opposite obtains. In the one, 
stimulation appears to be associated with increase, in the other, with de- 
crease in water content. In both, however, as pointed out above, it ap- 
pears to be associated with decrease in polarization. 

In general, it may be said that a galvanic current usually induces in 
the lower organisms chemical and physical changes which differ at op- 



332 MOTOR RESPONSES 

posite sides, the one facing the anode, the other the cathode; and that 
either or both of these two sets of changes may result in what is usually 
called a response, although not necessarily an orienting response. Thus 
while the motor responses in these organisms usually occur at the catho- 
dal side when the circuit is closed, Kiihne (1864), Verworn (1895), 
and McClendon (1911) observed in Amoeba and other rhizopods con- 
traction at the anodal side. In some of these organisms, the anodal con- 
traction appears to be involved in streaming toward the cathode; but 
there are other anodal responses which obviously have nothing to do with 
locomotion. For example, Loeb and Budgett (1897) assert that there is, in 
Amhlystoma, copious secretion of mucus on the anodal side. Moore 
(1926) obtained bioluminescence and contraction on the anodal side of 
the Ctenophores, Mnemiopsis, and Beroe. Lyon (1923) and Lund and 
Logan (1925) observed, in Noctiluca, a sort of contraction first at the 
anodal side and later also at the cathodal side, and sometimes the reverse. 

In all of the organisms referred to above, except Noctiluca, the 
anodal responses differ radically from the cathodal responses. This is 
very evident from the results of observations on Amoeba, in which it 
can be clearly seen that, after the circuit is closed, there is first local 
liquefaction of the plasmagel on the cathodal side, then contraction, 
and finally cytolysis on the anodal side. In Volvox, however, the response 
on the cathodal side in colonies which are positive to the cathode, is, in 
all perceptible characteristics, precisely the same as the response on the 
anodal side in those which are positive to the anode. Here, then, is an 
actual reversal in the action of the current, i.e., an actual reversal of 
Pfliiger's law. Closing the circuit apparently produces the same effect 
on the anodal side of colonies in certain physiological states as it does 
on the cathodal side of colonies in other physiological states. 

These physiological states are, as set forth above, specifically associated 
with those involved in reversal in the direction of photic orientation. As 
demonstrated above, these are dependent upon illumination, tempera- 
ture, and chemicals in the environment, which apparently control the 
electric charge carried by the colonies and the bodies within them. What 
is much needed now is a more comprehensive study of these charges, 
in relation to the chemical and the physical content of the environment 
as well as the character of the responses of the colonies. 



MOTOR RESPONSES 333 

Responses to Chemicals 
a. rhizopods 

None of the rhizopods except Aiiweha has been studied with refer- 
ence to motor responses to chemicals. Many of the observations made 
on Amoeba are so indefinite that further work under more carefully con- 
trolled conditions is highly desirable. In this work the large form, 
Pelomyxa carolinensis, which can now be readily procured, would doubt- 
less be very favorable. 

Using a capillary pipette, Edwards (1923) applied various chemicals 
locally to the surface of active specimens of A. proteus. With a few of 
these chemicals he obtained fairly definite results, which lead to the 
following conclusions: 

If an alkali comes in contact with the side of an active amoeba, stream- 
ing stops and a local protuberance is formed at the point of contact. If 
the solution is weak, the protuberance develops into a normal pseudopod, 
which continues indefinitely toward the source of the solution. If it is 
strong, the protuberance breaks at the tip and the central portion of the 
amoeba flows out, leaving nothing but a crumpled membrane. 

If an acid is applied, streaming stops and a similar protuberance is 
formed but it does not become large and does not develop into a nor- 
mal pseudopod. If the acid is weak, the streaming soon begins again 
and the protuberance gradually disappears. If it is strong, the protuber- 
ance is very small and pseudopods form on the region opposite the point 
of application. 

If acid is applied after a rupture in the surface of an amoeba has been 
produced by local application of an alkali and after the central portion 
begins to flow out, the flow immediately stops and the amoeba soon 
proceeds normally. These and other facts show that alkalies cause the 
cytoplasm of Amoeba to solate and that strong acids cause it to gelate; 
but what is involved in the formation of a protuberance by weak acid 
at the region of application is not clear. 

A strong solution of sodium chloride results in formation of pseudo- 
pods opposite the region of application; a weak solution results only in 
the formation of a protuberance in the region to which it is applied. 
Thus Amoeba is negative to a solution of this salt if it is strong, and 
positive if it is weak. This indicates that the former induces gelation of 



334 



MOTOR RESPONSES 



the cytoplasm, and the latter, solation. Whether or not this obtains for 
other salts has not been ascertained. 

Strong alcohol produces' a blister at the point of application, followed 
by formation of pseudopods on the opposite surface. 

The effect of all these substances is correlated with the kind of chemi- 




Figure 119. The relation between rate of locomotion, gel/sol ratio, and hydrogen-ion 
concentration in a balanced salt solution. A, Ai, rate of locomotion and gel/sol ratio in 
solutions containing salts in concentrations given in Table 2 ; B, Bi, rate of locomotion 
and gel/sol ratio in salts in concentration five times as great as those given in Table 2. 
Curve A, mean rates of movement of images of amoebae given in Table 2 ; B, means 
of the rates of movement of seventeen to twenty-four individuals for from five to seven 
minutes each in each hydrogen-ion concentration; Ai and Bj, mean gel/sol ratios cal- 
culated from results obtained in measurements made on seventeen to twenty-four indi- 
viduals in each hydrogen-ion concentration as described above. (After Pitts and Mast, 
1933.) 

cals, with their concentration in the medium surrounding the amoebae, 
and with the length of exposure to them. 

If these conclusions are valid, one would expect amoebae in a culture 
medium to aggregate in regions which are alkaline or which contain 
relatively little salt. Hopkins (1928) made some observations concern- 
ing the former. He put specimens into a drop of solution which was 



MOTOR RESPONSES 



335 



pH 7.1 and then joined this drop with other drops which were respec- 
tively pH 6 and 8. He found that the amoebae at the border between 
the drops formed pseudopods which protruded toward the drop added, 
no matter whether it was alkahne or acid. This indicates that amoebae in 




Figure 120. The relation between rate of locomotion, gel/sol ratio, hydrogen-ion con- 
centration, and sodium-ion concentration. Each of the curves represents a series of ex- 
periments conducted at constant sodium-ion concentration. The four solid curves are 
based on the measurement of the rate of locomotion of an average of 14.8 different 
specimens for an average of 88.1 minutes in each hydrogen-ion concentration tested. 
The two broken curves are based on the measurement of the gel/sol ratio in an average 
of 19 different individuals in each hydrogen-ion concentration tested. (After Pitts and 
Mast, 1934b.) 

a neutral solution tend to aggregate, either in acid or alkaline regions. 
The observations made were, however, not extensive enough to war- 
rant any definite conclusions. There is, then, nothing definite known 
concerning the relation between differences in the chemical composition 
of the medium and aggregation in any of the rhizopods, and but little 
concerning the action of chemicals in relation to changes in direction of 



336 



MOTOR RESPONSES 



movement. On the other hand, there are some very definite results con- 
cerning the relation between the rate of locomotion and the chemicals in 
the surrounding medium, especially hydrogen ions. 

Rate of locomotion and H-ion concentration. — Hopkins (1928) ob- 
served that A. proteus, in an ordinary hay culture fluid, is inactive if the 
fluid is neutral, but active if the fluid is either acid or alkaline, and that 
the rate of locomotion is maximum and nearly equal at about pH 6.5 



Z60 















y 


K^ 








1 


^' 






J 


,--^ 


»^ 


V 


" "^ 


&~ 


^, 




< 






C 


>'.-^ 


( 


r 




-^ 




f 




i 


> 


y 


/ 


< 


^^' 




■^ *. 




I 


/ 


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/ 


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Loiy 


)motio 


n 




^ 


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ooom 

• OOS 




, 
















9 

• 


OOltl 
005 








ptl 

Figure 121. The relation between rate of locomotion, gel/sol ratio, hydrogen-ion con- 
centration, and calcium-ion concentration. Curve O, mean rate of locomotion in 0.001 M, 
based on measurements on an average of 22.1 different specimens for 11.6 minutes in 
each hydrogen-ion concentration tested; curve #, mean rate of locomotion in 0.005 M, 
based on an average of 18.7 different specimens for a total average of 96.6 minutes in 
each hydrogen-ion concentration tested; curve 3, mean gel/sol ratio in 0.001 M, based 
on measurements on an average of 21.3 different specimens in each hydrogen-ion con- 
centration tested; curve O, mean gel/sol ratio in 0.005 M, based on measurements on 
a total average of 14.6 different specimens in each hydrogen-ion concentration tested. 
(After Pitts and Mast, 1934b.) 

and pH 8 respectively. Mast and Prosser (1932) confirmed these results. 
Pitts and Mast (1933), in a much more thorough study on A. proteus, 
demonstrated that the inactivity at neutrality is correlated with the rela- 
tion between the amount of sodium or potassium and calcium present; 
and that the relation between activity and the concentration of hydrogen 
ions varies greatly with the kind, the concentration, and the proportion 
of salts in the surrounding medium. Some of the results obtained in this 
study are presented in figures 119, 120, 121, and 122. These figures 
show the following: 



MOTOR RESPONSES 



337 



In a balanced salt solution the activity is minimum at neutrality and 
maximum on either side; but the activity at any given hydrogen-ion 
concentration varies with the salt concentration (Fig. 119). In sodium 
or calcium salt solutions (Table 2) the maximum rate of locomotion 
is nearly as high as in a balanced salt solution. Furthermore, the rate at 




Figure 122. The effect of adding calcium in different concentrations to 0.005 N sodium 
solutions, on the relation between hydrogen-ion concentration, rate of locomotion, and 
gel/sol ratio. The solid curve for the solutions containing calcium 0.001 M is based 
on measurements of the rate of locomotion, on an average of 14.75 different specimens 
for a total average of 89.9 minutes; that for calcium 0.0005 M, on an average of 18.6 
different specimens for a total average of 95 minutes; and that for calcium 0.001 M, on 
an average of 20.5 different specimens for a total average of 104.6 minutes in each 
hydrogen-ion concentration tested. The broken curve for the solution containing cal- 
cium 0.001 M is based on measurement of the gel/sol ratio, on an average of 19.8 
different specimens; that for calcium 0.0005 M, on an average of 17.9 different speci- 
mens; and that for calcium 0.0001 M, on an average of twenty different specimens in 
each hydrogen-ion concentration tested. (After Pitts and Mast, 1934c.) 



any given hydrogen-ion concentration varies with the salt concentration, 
although the relation between the rate and the hydrogen-ion concen- 
tration in the one differs greatly from that in the other, and there is no 
indication of inactivity at neutrality in either (Figs. 120, 121). If cal- 
cium salt is added to a solution of sodium salt, the activity decreases 
greatly at neutrality (Fig. 122). The ratio between the amount of plas- 
magel and the amount of plasmasol in Amoeba varies with the hydro- 
gen-ion concentration; but the rate of locomotion is not specifically cor- 
related with this ratio. 



338 MOTOR RESPONSES 

Table 2 : Rate of Locomotion of A. Proteus in Sodium and 
Calcium Salt Solution* 

Composition of the solution used to ascertain the effect of hydrogen-ion concentration 
on rate of locomotion and gel/sol ratio in a balanced salt solution. To obtain different 
hydrogen-ion concentrations, the acid and the alkaline components were mixed in dif- 
ferent proportions. 

Note that the concentration of Na, K, Ca, and Mg remains constant, no matter what 
the proportion of the two components is. 

In the second series of experiments the concentration of all the salts was increased 
five times (after Pitts and Mast, 1933). 



Acid Component 



Alkaline Component 



Molar Ratio 



NaH2P04 0.00150N 

KH2PO4 o.oooioN 

CaH4(P04)2 O.oooioN 

MgCl2 0.00005N 



NaOH 0.00 1 50N 

KOH O.oooioN 

Ca(OH)2 o . 000 1 oN 

MgCU 0.00005N 



Na 60 

K 4 

Ca 2 

Mg I 



* After Pitts and Mast, 1933, by permission of the Journal of Cellular and Comparative Physiology, 
Wistar Institute of Anatomy and Biology. 

Mechanics of response. — Concerning the action of chemicals produc- 
ing these responses on Amoeba, Pitts and Mast make the following state- 
ments : 

It is obvious that a substance in the environment may influence processes 
which occur in a cell either by entering the cell and acting directly on 
substances in the cell, or by acting on the surface of the cell in such a way 
as to retard or facilitate the passage into or out of the cell of other sub- 
stances which, owing to their presence or absence, induce alterations in 
internal processes. . . . 

It may be assumed, then, either (1) that the gel/sol ratio depends upon 
the entrance of salts into the cell and reaction between these and internal 
substances, and that the rate of entrance of salts varies with the hydrogen 
ion concentration; or (2) that the gel/sol ratio varies with the rate of en- 
trance of hydrogen and hydroxyl ions into the cells and reaction between 
these and internal substances, and that the rate of entrance of these ions 
varies with the concentration of the salts and the hydrogen ions; or (3) that 
the gel/sol ratio depends upon the exit of substances from the cell, e.g., 
water, and that this depends upon the hydrogen ion concentration, and the 
salt concentration and the kinds of salts present. Let us now attempt to 
ascertain if the processes in question are in accord with any of these groups 
of assumptions. . . . 

If they are in accord with the first of these assumptions, the gel/soI 
ratio must vary directly or indirectly with the amount of salt that enters the 
cell and this must vary directly or indirectly with the hydrogen ion con- 



MOTOR RESPONSES 339 

centration. If it varies indirectly with the amount of salt that enters and 
this varies either directly or indirectly with the hydrogen ion concentra- 
tion practically none of the results obtained are in accord with the assump- 
tions. If it varies directly with the amount of salt that enters and this varies 
directly with the hydrogen ion concentration the results obtained with 
balanced salt solutions and some others are in full accord with the assump- 
tions; but the assumption that the entrance of salts varies directly with 
hydrogen ion concentration is not in harmony with the results obtained by 
practically all who have investigated this problem. Moreover, the assump- 
tions do not account for the independence or inverse variation between hydro- 
gen ion concentration and gel/sol ratio in calcium solutions. . . . 

If they are in accord with the second of the three assumptions made above, 
the gel/sol ratio must depend upon the hydrogen ion concentration with- 
in the cell and this must vary with the concentration of the salts and the 
hydrogen ions in the surrounding medium. If it varies indirectly with the 
hydrogen ion concentration of the surrounding medium and directly or 
indirectly with the salt concentration, few if any of the results obtained 
are in accord with the assumptions. If it varies directly with the hydrogen 
ion concentration and the salt concentration of the surrounding medium, 
the results obtained with balanced salt solutions and some others are in ac- 
cord with the assumptions, but those obtained with calcium solutions are 
not. There is, moreover, no evidence which indicates that the hydrogen ion 
concentration within Amoeba varies appreciably with variation in the hydro- 
gen ion and the salt concentrations of the surrounding medium (Chambers, 
1928). ... 

In accord with the third group of assumptions, the gel/sol ratio must vary 
with the rate of exit of substances from the cell, and this must vary di- 
rectly or indirectly with the salt and the hydrogen ion concentration, and it 
must also vary with the kind of salts present in the environment. Without 
entering upon a detailed analysis of the correlation between these assump- 
tions and the results under consideration, it is evident that, no matter what 
combination is selected, there are between them and the results inconsist- 
encies of the same nature as those presented above. . . . 

It is consequently obvious that the results in hand cannot be consistently 
explained by any one of the three groups of assumptions made, and that 
there must be a fairly complicated interaction between the various factors 
involved. If this is true, the statement, without qualification, that any given 
factor facilitates gelation or solation is obviously so incomplete that it is 
without value. This conclusion is in full harmony with that reached by 
Mast and Prosser (1932). . . . 

In reference to the relations between rate of locomotion and kind of 
salts, salt concentration and hydrogen ion concentration, the results ob- 
tained are, on the basis of any one of the groups of assumptions considered 



340 MOTOR RESPONSES 

above, even less explicable than are those in reference to the gel/sol ratio. 
The outstanding difficulty here concerns the remarkable decrease in rate 
of locomotion as neutrality is approached either from the acid or from 
the alkaline side. The results presented show that this decrease occurs in 
balanced solutions; that is that it is specifically correlated with the Na/Ca 
ratio; that the higher this ratio within the limits of the concentrations 
tested, the greater the decrease; and that it does not occur in solutions con- 
taining only one salt. . . . 

The results show that if calcium is added to a solution containing only 
sodium salts, the rate of locomotion in the alkaline range increases greatly, 
with but little change in the acid range and in the region of neutrality ; and 
that if sodium is added to solutions containing calcium salts, the rate de- 
creases greatly in the region of neutrality, with but little change else- 
where. . . . 

The questions now arise as to why addition of calcium to solutions con- 
taining only sodium salts causes great increase in rate of locomotion in the 
alkaline range, and why addition of sodium to solutions containing only 
calcium salts causes great decrease in the rate in the region of neutrality. 

Similar questions have arisen in reference to the bimodal curves obtained 
by a number of other investigators in plotting the rate of various physiological 
processes against hydrogen ion concentration, e.g., by Robbins (1926) and 
Farr (1928) in various processes in plants; by Ephrussi and Neukomm 

(1927) in the resistance to heat in the eggs of a sea urchin; by Hopkins 

(1928) in the rate of locomotion in A>?ioeba; by Mast (1928) in the rate of 
assumption of stellate forms in Amoeba; by Eisenberg-Hamburg (1929) in 
the rate of increase in water content in infusoria; by Chalkley (1929) in 
water content and gel/sol ratio in Amoeba; and (1930a, 1930b) thermal 
death rate in Paravieciiim ; by Chase and Glaser (1930) in rate of locomo- 
tion in Paramecium : and by Mast and Prosser (1932) in rate of locomotion 
in Amoeba. . . . 

Only a few of these investigators attempted to elucidate the phenomenon. 
Mast and Prosser (1932), as previously stated, concluded that it is cor- 
related with salt concentration. We have already considered this view. 
Robbins (1926) contends that the hydrogen ion concentration, at which the 
median minimum in the plant processes studied occurs, coincides with the 
isoelectric point of the principal proteins in the plant. Farr (1928) main- 
tains, however, that this view is not tenable. He found in observations on 
the relation between hydrogen ion concentration and rate of growth in root 
hairs of collards that the hydrogen ion concentration at which the median 
minimum occurs varies greatly with salt concentration and he concludes that 
it therefore cannot be specifically correlated with the isoelectric point of 
any given protein in the organism. . . . 

In reference to Amoeba, the constancy of a median minimum rate of 



MOTOR RESPONSES 341 

locomotion at pH 7.0 in various solutions indicates some fixity of mechanism 
determining this median minimum. This mechanism might be correlated 
with the behavior of the membrane in the neighborhood of an ampholyte 
isoelectric point near neutrality in accord with the view of Robbins (1926). 
But arguing against such an isoelectric point are the pertinent facts that, 
(1) the median minimum is lacking in solutions of single salts, though 
locomotion in the dilute solutions occurs at hydrogen ion concentrations in 
which it is usually found; (2) the difference between the maximum and 
the median minimum rate of locomotion depends on the sodium/calcium 
ratio and only slightly if at all on the total salt concentration; (3) the 
cations have marked effect on the rate of locomotion on the acid side of 
the neutral point as well as on the alkaline side; (4) the anions (so far 
as chloride and phosphate are concerned) have little effect down as far 
in the acid range as the observations were made. . . . 

We are at present unable to suggest a satisfactory explanation for this 
median minimum. Whatever the cause of it may be, the relation between 
the rate of locomotion in Amoeba and the factors in its environment is doubt- 
less fairly complex, for it is probable that these factors influence locomotion 
in it by their action on the surface, affecting adhesiveness and other prop- 
erties of the surface, as well as by their action on permeability of the sur- 
face membrane (Mast, 1926a). 

B. MARINE AMOEBAE • 

Pantin (1923-31) made observations on the relation between the rate 
of locomotion in a marine amoeba and various chemicals. He found that 
as the hydrogen-ion concentration increases, the rate increases rapidly 
from zero at pH 10 to a maximum at about pH 8, and then decreases 
rapidly to zero at pH 5.5. He observed no indication of decrease in 
activity at neutrality. He maintains that the rate is closely correlated with 
the relative concentration of sodium, potassium, magnesium, and cal- 
cium salts, that more than one of these salts is necessary for locomotion, 
and that calcium is required in all combinations. He maintains that cal- 
cium functions primarily in the contractile mechanism, and the others in 
the regulation of permeability. The evidence presented in support of 
these conclusions is, however, not unequivocal. 

Oxygen is necessary for locomotion in Amoeba (Hulpieu, 1930; Pan- 
tin, 1930) but only in very low concentrations. Pantin maintains that 
none is immediately necessary, and that it functions in recovery some- 
what as it does in the contraction of muscles. This, however, has not been 
demonstrated. 



342 MOTOR RESPONSES 

C. CILIATES 

The most prominent response of the cihates to chemicals consists in 
reversal in the direction of the effective stroke of the locomotor cilia, 
and consequent backward swimming. This reversal may be so brief that 
it results in scarcely perceptible backward movement, or it may continue 
for several minutes. It has been investigated in some detail in Paramecium, 
but not in any of the other ciliates. The rate of locomotion in all the 
ciliates is doubtless correlated with the chemical composition of the 
surrounding medium, but no measurements concerning this correlation 
have been made in any of them. 

The reversal in the direction of the stroke of the cilia of paramecia 
in response to chemicals extends to the entire surface of the body, ex- 
cept the oral groove. It consequently results in backward swimming. It 
is the same as the reversal induced by contact and by rapid changes in 
temperature or osmotic concentration, and it is usually followed by turn- 
ing toward the aboral surface and forward movement in a new direction, 
i.e., it usually constitutes the first stage in the avoiding reaction. 

Jennings (1906) describes in detail how this response results in aggre- 
gation of paramecia in regions which contain ineffective chemicals in re- 
lation to those in adjoining regions, e.g., regions which are slightly 
acid, surrounded by regions which are slightly alkaline. If this obtains, 
he says, paramecia do not respond as they enter the acid region, but do 
when they reach the edge of this region and are about to enter the alkaline 
region. They consequently remain in the acid region. As more paramecia 
enter, owing to random movements, an aggregation is formed. 

He maintains that these responses usually result in aggregations in re- 
gions which are favorable for the organisms, but that there are exceptions. 
That is, he holds that these responses are, in general, adaptive. However, 
he has made no suggestions concerning the processes in the organism in- 
volved in producing these responses. 

Merton (1923) found that sodium and potassium salts induce reversal 
in Stentor and that calcium and magnesium do not, but he offers no 
suggestions concerning the nature of the action of the former nor the 
cause of inaction of the latter. 

Mast and Nadler (1926) ascertained the effect of fifty-six different 
chemicals on the direction of the effective stroke of the cilia in Parame- 
cium. They maintain that all of the monovalent cation salts and hydrates 



MOTOR RESPONSES 343 

tested (thirty-one), except (NH,)oSO, and NH.CaHgO,, induce rever- 
sal; but that none of the bivalent and trivalent cation salts tested (nine- 
teen), except CaHPO, and MgHPO,, induce it. Also that Ba(OH)2, 
H3PO4 and HXoO^ induce reversal, while HCL and lactose do not. 

These authors contend that the duration of reversed action is closely 
correlated with the concentration of the salts; that it varies with the kind 
of salt at any given concentration; and that bivalent and trivalent cation 
salts neutralize the effect of monovalent salts. 

Concerning the physiological processes involved in reversal in ciliary 
action, they make the following statement: 

Copeland (1919, 1922) and Grave and Schmitt (1925) demonstrated 
that the cilia in higher forms are closely connected with nerve fibers and that 
their action is in all probability controlled by nerve impulses. The results 
obtained in the investigations of Yocum (1918), Taylor (1920), Rees 
(1922), Visscher (1927) and others on the neuromotor apparatus in the 
protozoa, indicate that the action of the cilia in these forms is similarly con- 
trolled. If this is true, the question arises as to how environmental changes 
which have been observed to induce reversal in ciliary action influence 
the neuromotor apparatus. This may be conceived to be either through 
chemical changes produced in the receptors or elsewhere in the organism 
or through changes produced in the electric potential at the surface, or in 
the permeability and the consistency of the surface layer. . . . 

The results obtained by Mast and Nadler indicate that reversal in ciliary 
action is largely dependent upon the cations, that it is induced by monovalent 
but usually not by bi- and tri-valent cation salts, and that it depends upon the 
concentration of the salts. . . . 

It is well known that adsorption of the cations is usually relatively greater 
in bi- and tri-valent than in monovalent cation salt solutions and that the 
adsorption varies with the concentration of the salts in the solution. This 
seems to indicate that the difference in the action of the monovalent and 
the bi- and tri-valent cation salts and the difference in the action of different 
concentrations of the monovalent cation salts is at least in part associated 
with differential adsorption of the ions in the various solutions, resulting 
in changes in the electrical potential at the surface of the paramecia, which 
directly or indirectly produce impulses in the neuromotor apparatus which 
pass to the cilia and influence their action. The facts, however, that Ba(OH)2 
produces reversal while BaCl^, CaCl,, MgCL do not, that H^QO^ and 
H3PO4 produce reversal while HCl does not, that CaHPO^ and MgHPO^ 
produce reversal while Ca3(PO_j)2 and Mg(POJo do not, all indicate 
that there must be other factors involved in reversal aside from differential 
adsorption of the cations. 



344 MOTOR RESPONSES 

Oliphant (1938) made a much more extensive and thoroughly con- 
trolled study of the effect of monovalent and bivalent cation salts on 
ciliary reversal in ParameciutJi. 

The results obtained support the contentions of Mast and Nadler 
(I926) that the monovalent salts induce reversal, whereas bivalent ca- 
tion salts do not, and that the duration of the reversed action varies 
with the kind and the concentration of the salts; but they do not support 
their contentions as to the nature of this variation. The results show that 
the effect of the salts is due primarily to the action of the cations, and 
that the anions have little, if any, effect. They show that the order of 
effectiveness of the cations is K>Li>Na>NH^, and that the duration 
of their effect varies inversely with the temperature. 

Oliphant (1938) cites work which indicates that in Paramecium, 
Amoeba, Actinos phaevmm , Spirogyra, root hair of Trianea, and eggs of 
Arhacia, monovalent cations induce increase, and that bivalent cations 
decrease the viscosity of the cytoplasm (Spek, 1921; Heilbrunn, 1923, 
1931, Cholodnyj, 1923; Weber, 1924). He concludes that this indicates 
that reversal in ciliary action is correlated with increase in viscosity of 
the cytoplasm, and contends that this conclusion is supported by the fact 
that "reversal in response to temperature occurs only at temperatures 
almost immediately lethal," i.e., at temperatures which cause marked 
increase in viscosity. He holds that the action of the cilia is controlled 
by the neuromotor apparatus and that increase in viscosity produces im- 
pulses in this structure which cause reversal in ciliary action, but he thinks 
that changes in electric potential, in permeability, in the consistency of 
the surface layer, or in the chemical composition of the receptors or 
other structures in the organism may be involved. 

It is obvious from the above discussion that there is still much to 
be learned concerning the processes involved in the responses of the 
ciliates to chemicals. 

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348 MOTOR RESPONSES 

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MOTOR RESPONSES 349 

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350 MOTOR RESPENSES 

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MOTOR RESPONSES 351 

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CHAPTER VI 

RESPIRATORY METABOLISM 

Theodore Louis Jahn 

Studies of the respiration of the Protozoa have, for the most part, 
been fragmentary, and our information on the subject resembles an ac- 
cumulation of a number of more or less isolated data, rather than a 
unified body of knowledge. This situation is due to a variety of causes, 
chief among which is probably the fact that most studies of respiration 
have been made by physiologists who chose, among the members of the 
animal kingdom, the organisms which seemed to be the most suitable 
for a particular type of experiment. From this viewpoint the study of 
protozoan respiration has suffered a severe handicap, in that a consider- 
able mass of protozoan protoplasm, free from bacteria and other organ- 
isms, has not always been easy to obtain, and in that our methods for 
measuring very small rates of respiratory exchange have not been nearly 
as accurate or as convenient as we might desire. However, with gradual 
technical advances, it seems probable that in the near future we shall see 
the development of an organized account of protozoan respiration, and 
it also appears probable that this development will take place among 
investigators who are primarily interested in the Protozoa. Therefore, 
it seems advisable to combine a review of data on protozoan respiration 
with a discussion of the general problems of respiratory metabolism in 
other biological materials, and to outline for the student not thoroughly 
trained in the lore of respirometry some of the purposes, methods, and 
possible interpretations of such a study. 

Among the Protozoa, the intake of oxygen does not require compli- 
cated respiratory mechanisms. Apparently diffusion, high rate of water 
exchange, and protoplasmic movements (cyclosis, amoeboid streaming, 
and "metabolic" movements) are sufficient to maintain a suitable level 
of O2 tension in the protoplasm and to prevent the accumulation of toxic 
amounts of CO2. The mechanisms which are responsible for protoplasmic 
movements and the high rate of water exchange are more properly treated 



RESPIRATORY METABOLISM 353 

under the subjects of movement, permeability, and excretion. Therefore 
the problem of respiratory metabolism of a protozoan organism, at least 
for the present discussion, is easily resolved into a problem comparable 
to that of cellular respiration in the Metazoa. 

Purposes of Studying Respiration 

One of the first questions to be considered is: "What can be learned 
by studying respiration?" The measurement of gaseous exchange is not 
an end in itself, but it is a tool which, when used singly or in combina- 
tion with other tools, may help us to obtain answers to the following 
types of questions. 

1. What is the rate of energy expenditure of the organism.-' How does 
the metabolic rate (basal and otherwise) of one species compare with 
that of another.^ How does it change during starvation? Or conjugation? 
Is this rate dependent upon the Oo or CO^ tension of the environment? 
How is it affected by narcotics? How does it vary with temperature? Or 
with other chemical and physical factors of the normal environment? 

2. What is the source of this energy? Is it obtained by oxidation of 
fats, carbohydrates, or proteins? Or by anaerobic oxido-reductions? Can 
this source be shifted by changing the chemical or physical environment 
of the organism? What are the intermediate products formed during 
oxidation of the substrate? Is oxidation of the substrate complete (i.e., 
to COo and H.O), or may these intermediate products be excreted by 
the cell? 

3. What is the mechanism by which energy is obtained from the sub- 
states? Is this brought about through the intermediary action of dehydro- 
genase, cytochrome, and Warburg's respiratory enzyme? Or is it brought 
about through dehydrogenase and reversible oxidation-reduction systems, 
such as yellow pigment or pyocyanine? Or by glutathione? Can these 
substances be replaced by artificial oxidation-reduction systems? Can a 
shift in this mechanism of respiration be induced by changing the avail- 
able substrate? How is this mechanism related to the degree of anaerobio- 
sis which the organisms can endure? Is the organism capable of synthesiz- 
ing the respiratory enzymes from simple substances, or must they be 
obtained from complex outside sources, i.e., from vitamins? 

Measurements of the respiratory metabolism of an organism may be 
used as indices of the rate at which it uses energy, the substrate from 



354 



RESPIRATORY METABOLISM 



which this energy is derived, and the mechanism by which it is obtained 
from the substrate. In addition to these factors, there is a possibihty of 
a direct relationship between certain metabohc processes and the patho- 
genicity of parasitic forms. 

Methods of Measuring Aerobic Respiration 

Methods that have been or might be used for measurement of aerobic 
protozoan respiration fall naturally into two groups — those applicable 



Table 3: Sensitivity of Respirometers 



Type of Respirometer 


T^earest Unit to 

Which Meniscus 

Can Be Read* 


Approximate Sensitivity 

in Terms of Scale 

Divisions 


Standard Warburg 
(Warburg, 1926) 




0.2 mm. 


I mm. = 1.0-2.0 mm.^ O2 


MicrosemidifFerential 
(Duryee, 1936) 




Q. 2 mm. 


I mm. = o. 5 mm.' O2 


MicrodifFerential 
(Fenn, 1928) 




Q. I mm. 


I mm. = 0.3 mm.3 O2 


MicrodifFerential 
(Described in text) 




Q.2 mm. 


I mm. = 0.2 mm.' O2 


Straight capillary tubes 

(Howland and Bernstein, 193 1) 


Q.oi mm. 


o.oi mm. = 0.001 mm.' 0> 


Straight capillary tubes in closed air 
chamber 
(Gerard and Hartline, 193,4) 


.006 mm. 



0.006 mm. = 0.0013 mm.' O2 


Microdifferential 
(Schmitt, 1933) 




micron 


I micron= 0.0005 mm.' O2 


Cartesian diver 

(Needham and Boell, 


1938) 


0.2 ^'"^ 


I mm. = 0.001 mm.' O2 



* It is assumed that the meniscus is read to the nearest 0.2 mm., unless otherwise stated by the 
authors. In those cases where the author claims greater reading accuracy, the smallest unit of change 
which can be detected is given in this column. If stability is adequate, the sensitivity of several models 
may be considerably increased by the use of special reading devices. 

to concentrated suspensions of organisms, and those applicable to a few 
or to single cells. For concentrated suspensions, titration, gas analysis, 
and standard manometer methods have been used; and for studies of 



RESPIRATORY METABOLISM 355 

single protozoan cells, micromanometric methods have been devised 
(Kalmus, 1927; Rowland and Bernstein, 1931). More recently, still 
better micromanometric methods have come into existence, but these have 
not yet been applied to the respiration of protozoa. Table 3 gives the 
sensitivity of various types of manometers, some of which have not yet 
been used to measure the respiration of Protozoa. It should be remem- 
bered that whenever a respirometer is made more sensitive to changes 
in gas volume produced by organisms, it simultaneously becomes more 
sensitive to slight changes produced by the environment (thermal and 
barometric effects) and to inaccuracies arising from imperfect design 
and construction (ground-glass connections and stopcocks, surface phe- 
nomena at the meniscus of manometer fluid, inaccuracies of capillary bore 
and so forth). Therefore, stability of the apparatus, on which final ac- 
curacy must depend, becomes more and more difficult to obtain. For that 
reason the most sensitive types should be reserved solely for those prob- 
lems in which concentrated suspensions are undesirable or unobtainable. 
By comparison of Tables 3 and 4, it should be possible to determine ap- 
proximately the type of respirometer necessary for any one of a variety 
of problems. 

1. TITRATION METHODS 

a. Dissolved O^ determinations. For any aquatic animal it is possible 
to measure Oo consumption by placing the organisms in a closed chamber 
filled with water of known 0„ content and by measuring the amount of 
O2 left after a definite period of time. For this purpose a modified Win- 
kler titration method is usually used {Standard Methods of Water Analy- 
sis, 1936). Special precautions are necessary whenever the animals can- 
not be removed from the solution, or if iron is present. This method has 
been used by Lund (1918a, 1918b, 1918c) and Leichsenrmg (1925) 
on Paramecium and Colpoda. 

b. Measurement of CO2 production. Production of CO2 may be 
measured by placing the organisms in a small amount of solution in a 
small open container. This is placed inside of a larger closed container 
in which an alkali, preferably Ba(OH)2, is present. The COo given off 
by the organisms is absorbed by the alkali, which can then be titrated 
with acid in the presence of an indicator. This method was recom- 
mended by Lund (1918d) for use with Paramecium. 



356 RESPIRATORY METABOLISM 

The above titration methods are applicable only to quite large num- 
bers of organisms. It seems possible that these methods could be im- 
proved by the use of accurately controllable microburettes, smaller vol- 
umes of liquid, and so forth, but it is doubtful whether they could 
be made as accurate and reliable as some of the manometric procedures 
discussed below. Also, respiratory quotients are not easily obtained by 
titration methods. 

2. GAS ANALYSIS 

Soule (1925), Amberson (1928), and Root (1930) have applied the 
standard Haldane-Henderson methods of gas analysis to respiration of 
the Protozoa. These methods are adequate for use with rather con- 
centrated suspensions and pressures, and possess certain definite ad- 
vantages when Oo or especially CO, tension is being varied experimen- 
tally and would have to be determined separately if manometric methods 
were used. Whenever gases other than COo are evolved by an organism, 
gas analysis seems to be the only satisfactory method of measurement. 
The details of gas analysis methods are discussed by Peters and van 
Slyke (1932). Soule (1925), for studies of the metabolism of Leish- 
mania tropica and Trypanosoma leivisi, used gas analysis, supplemented 
by readings of an insensitive manometer, the purpose of which was 
principally to indicate when gas exchange was taking place (method 
described in detail by Novy, Roehm, and Soule, 1925). 

3. STANDARD MANOMETRIC METHODS 

The principle of the manometric method is somewhat as follows: 
the organisms, in a suitable immersion medium, are placed in a closed 
flask large enough so that a considerable air space is present. The flask 
is connected to a capillary manometer tube partially filled with a liquid. 
Alkali may be present in a separate small container inside of the flask. 
If so, then CO, is absorbed, and the amount of Og consumed may be 
measured by means of the movement of fluid in the manometer tube 
as changes in volume (Haldane, Thunberg, Winterstein, Duryee, and 
Dixon types), or in pressure at a given volume (Warburg), or as the 
resultant of simultaneous changes in both (Barcroft difl^erential type). 
Manometric methods, although very simple in outline, are filled with 
pitfalls for the inexperienced investigator, and a careful reading of the 



RESPIRATORY METABOLISM 357 

excellent treatise of Dixon (1934) is recommended. In this publication 
the theory and the more common forms of the apparatus are described 
in detail. 

The Barcroft differential type can be made sufficiently small and 
sensitive for the study of respiration of Protozoa, when moderately con- 
centrated mass cultures are available. A manometer designed by Dr. 
T. C. Evans (modified from that of Bodine and Orr, 1925), which 
has been in use in this laboratory for some time, seems to be quite 
suitable. It resembles the standard Barcroft (Dixon, 1934, p. 37) ex- 
cept that the two stopcocks are replaced by one double stopcock, which 
insures the simultaneous opening and closing of both flasks. The cups 
may be relatively small (about 5 cc), and the U-shaped portion of 
the capillary (0.3 mm. bore) is placed at an angle of about twenty 
degrees from the horizontal. This design combines a high degree of 
sensitivity with ruggedness and dependability. The Duryee ( 1936) modi- 
fication of the Thunberg-Winterstein principle and the Fenn (1928) 
form of microdifferential respirometer also seem to have a sensitivity 
adequate for moderately concentrated suspensions. Schmitt (1933) has 
devised an extremely sensitive form of microdifferential manometer, in 
which the gain in sensitivity and accuracy is due chiefly to an elaborate 
reading device and a system of temperature control which is stated to 
make meniscus movements of as little as one micron both detectable and 
significant. 

4. CAPILLARY MANOMETER 

The use of capillary tubes for measuring respiration of single protozoan 
cells was introduced by Kalmus (1927, 1928a). This method was 
improved by Howland and Bernstein (1931), who by means of a micro- 
injection device drew small amounts of oil, air, KOH solution, and 
water containing an animal into small capillary tubes, so that they were 
finally arranged in the following order: oil, KOH, air, animal in water, 
oil. As the animal consumed O^, the distance between the oil-water and 
the oil-KOH interfaces decreased. This change was measured micro- 
scopically by means of an ocular micrometer and a calibrated mechanical 
stage. By using control tubes made in a similar manner but without an 
animal, it was possible to correct for slight movements due to thermo- 
barometric changes and to osmotic differences between the water and 



358 RESPIRATORY METABOLISM 

KOH. Gerard and Hartline (1934) have improved the method by 
enclosing the tubes in an air tight chamber to ehminate barometric dis- 
turbances, and by using a screw micrometer to increase accuracy of 
reading. 

5. CARTESIAN DIVER ULTRAMICROMANOMETER (NEEDHAM AND BOELL, 
1938) 

This is an appHcation of the principle of the Cartesian diver for use 
as a constant-volume manometer. The "diver" chambers are constructed 
from capillary tubing and consist of a bulb partially filled with gas, 
an open capillary neck, and a solid glass tail to ensure that the diver 
floats upright. The diver is placed in a closed chamber partially filled 
with a strong salt solution, the specific gravity of which is such that the 
diver maintains a position below the surface of the salt solution, and 
that a small amount of this solution enters the neck. If the amount of 
gas in the diver is changed by a reaction, more salt solution will be 
drawn in or forced out of the neck, the specific gravity of the diver 
will change, and the level of flotation will also change. By changing 
the pressure on the salt solution, the diver may be brought back to any 
given level. Therefore, the diver may serve as a constant-volume ma- 
nometer, and changes in the amount of gas in the chamber may be calcu- 
lated from the changes in the external pressure which are necessary to 
maintain the diver at a definite level. This application of the Cartesian 
diver was suggested by Linderstrom-Lang (1937), and has been used 
for parts of amphibian embryos by Needham and Boell (1938), who 
describe the use of this instrument for measurement of O2 consumption, 
anaerobic glycosis, and respiratory quotient. From Table 3 it may be 
noted that this instrument when read only to 0.2 mm. has a sensitivity 
as great as those which employ special reading devices, and that it 
therefore has the possibility of being made more sensitive. 

Aerobic Respiration 
1. the normal rate of respiration 

One fundamental essential in measuring the respiration of any bio- 
logical material is that other material, also capable of respiratory ac- 
tivity, be absent or very well controlled. This means that bacteria must be 
absent, or at least must contribute only a negligible amount to the 



RESPIRATORY METABOLISM 359 

measured respiration. Various workers have tried removing bacteria 
by washing and filtering, or have corrected the figures for O, consump- 
tion by running controls of bacteria without Protozoa. Data obtained 
by these methods are extremely difficult for a reviewer to evaluate 
critically, and usually one may either accept them at face value until 
they can be checked with bacteria-free cultures or ignore them entirely. 
In the present discussion the tendency has been to accept all data in 
which the magnitude of the error is not obviously large, and to point 
out possible difficulties involved. In view of the fact that some bacteria 
have a respiratory rate per gram many times that of other types of cells 
(the rate for Azotobacter is stated by Burk [1937] to be equivalent to 
that of a 200-pound man consuming one ton of glucose per hour), the 
present viewpoint may be considered far from conservative. In some 
cases (e.g., the papers on cyanide insensitivity of Parmnec'nan) the data 
on Protozoa seem quite adequate to prove the principal conclusions of 
the author, but are not accurate enough to afford detailed comparisons 
of respiratory rate. In such cases only the main points (e.g., insensitivity 
to cyanide) are given serious consideration. In only a few cases have 
investigators used bacteria-free cultures for measurement of respiration 
(cf. Table 4). 

For comparative purposes in work with metazoan tissues, it is custom- 
ary to express oxygen consumption in cubic millimeters (at normal tem- 
perature and pressure denoted as N.T.P.) per hour per milligram of dry 
weight of the tissue (symbolized by Qoa)- In the protozoan literature, 
where the rate of O, consumption is expressed in absolute units, this unit 
is sometimes the Q02 but is more usually mm^ per hour per organism, 
principally because the counting of organisms is simpler than measuring 
dry weight. Some authors use the symbol Q02 for Oo consumption per 
1,000,000 or per 100,000,000 organisms (e.g., von Fenyvessy and 
Reiner, 1928; Hall, 1938), but it seems preferable to avoid confusion 
by retaining this symbol for its original meaning and using a new symbol 
for consumption per 1,000,000 organisms, perhaps Q02 ^s used in Table 
4. However, since not dry weight, nor wet weight, nor number of or- 
ganisms affords the possibility of comparing the oxygen consumption per 
unit of respiring protoplasm, these discrepancies are not as important as 
one might at first suppose. In the case of flagellates such as Astasia and 
Chilomonas , in which a high percentage of the weight may be in the 



360 RESPIRATORY METABOLISM 

form of carbohydrate reserves, either dry or wet weight would be a poor 
index of the amount of respiring protoplasm. If one wishes to compare 
the rate per unit of protoplasm, the best index is probably the nitrogen 
content, because protein and other nitrogen-containing compounds are 
not ordinarily stored as reserve food. However, in the case of Protozoa 
which secrete nitrogen-containing tests, this criterion might also be 
very poor. Therefore any comparison of the absolute rate of different 
species, even after all differences in technique, immersion fluid, and 
physiological condition of the animal have been overcome, must usually 
be made with reservations, or at least with an adequate understanding of 
the limitations involved. Some authors have chosen to calculate O^ 
consumption in terms of cubic millimeters of organisms, but the errors in- 
herent in the methods of packing the animals for measurement (usually 
centrifuging) or in calculating volume from linear dimensions are too 
great to allow close comparison of data for different types of organisms. 
However, for an extended series of experiments on the same or very 
similar organisms, the use of dry weight (A. Lwoff, 1933) or of volume 
measurements (Elliott, 1939) seems to be entirely satisfactory. 

If concentrated suspensions of organisms are used, the rate of shaking 
should be carefully controlled. The importance of this factor is demon- 
strated by the data of Hall (1938). If ammonia is produced by an 
organism, it is necessary to maintain acid within the respiring chamber 
in order to obtain true values of O, consumption. This very important 
procedure is discussed by Specht (1935). 

Another question which arises in expressing results in absolute form 
is that of measuring basal metabolism, i.e., the metabolism of rest. In a 
mammal, for instance, there are certain well-defined limitations of condi- 
tions under which Oo consumption may be termed a measurement of 
basal metabolism. In a protozoan it is more difficult, if at all possible, 
to apply these criteria, and in all known measurements we have a sum 
of the total metabolic processes, i.e., of those to which we refer as "basal," 
those due to movement of the organism, and, if the medium is nutrient, 
those due to the manufacture of reserve food material and to growth. 
The energy expended for each of these purposes will probably vary 
with the species, the physiological state, and the environmental condi- 
tions. 

If the metabolic substrates of an organism undergo complete oxida- 



RESPIRATORY METABOLISM 361 

tion, we are able, by means of measurements of O, consumption, to 
determine directly the amount of energy available to the organism. If 
oxidation is incomplete, a further knowledge of the oxidation products 
is necessary. It is usually assumed, unless we have knowledge to the 
contrary, that oxidation is complete in the aerobic Metazoa (cf. intes- 
tinal nematodes, von Brand and Jahn, 1940). Among the bacteria and 
also among the Protozoa this is not always true, even in the presence of 
normal O, tension. However, since carbohydrate cleavage and intra- 
molecular oxidation, even in the presence of O2, may be considered an 
anaerobic process, that question will be discussed under anaerobiosis. 

From studies on the heat of combustion, we know that complete 
oxidation of glucose yields 677,000 calories per gram molecule, or about 
3,700 calories per gram. Complete combustion of protein yields 5,700 
calories per gram, and fats yield 8,000-9,000 calories per gram. There- 
fore, if we know the substrate being oxidized and the rate at which O, 
is consumed, we can calculate the energy made available by oxidation. 
According to the equation 

QHi^O, + 6 O2 -^ 6 CO. + 6 H2O + 677,000 cal. 

one gram molecule of glucose requires six gram molecules of O^. The 
volume of O, consumed (at N.T.P.) is 6 X 22.4 liters, or 134.4 liters. 
The ratio of O, consumed to calories released is 134 liters/677,000 
calories, or about one calorie for each 200 mm'' of O, consumed. Similar 
calculations may be made for fats and proteins. 

Table 4 contains most of the known data for respiratory rates for 
the Protozoa which can be expressed in absolute terms — either as mm^ 
O2 per organism per hour, or mm'^ O2 per gram dry or wet weight per 
hour. Similar tables are given by von Brand (1935) and Hall (1938). 

2. THE EFFECT OF O, TENSION ON O^ CONSUMPTION 

For many types of biological material it has been quite well established 
that, under usual experimental conditions, O, consumption is inde- 
pendent of O2 tension, within very wide limits (exceptions cited by 
Tang, 1933, McCoy, 1935). Recently, however, Kempner (1936, 1937) 
demonstrated that this is not true for several species of bacteria, for 
human leucemic leucocytes, for red blood cells of man, fowl, and alli- 
gator, and for pine needles if CO2 is present or if the temperature is 



362 



RESPIRATORY METABOLISM 



above 25° C. With these same materials in COa-free alkaHne media 
below 25° C, O, consumption was independent of Oo tension. The 
effect of Oo tension apparently varied with pH, COg tension, salt con- 

Table 4: Measurements of Protozoan Respiration 







Mm^ O2 










Mm^ O2 per 
Hour per 


per Hour 


Tem- 


Bac- 




Species 


Million 


per mg. Dry 
Weight 


perature, 
C. 


teria- 
free 


Investigator 




Qa= 


Q°. 




Paramecium caudatum 


120 (CO2) 




21° 


No 


Barratt (1905) 




140 




21° 


No 


Lund (1918c) 




2,250 




21" 


No 


Zweibaum (1921) 




3,900 




19° 


No 


Necheles (1924) 




5,600 




22° 


No 


Kalmus (1928b) 




500 




21.2° 


No 


Howland and Bern- 
stein (193 1) 


Paramecium multimicrO' 












nucleatum 


1,021 




25° 


No 


Mast, Pace, and Mast 
(1936) 


Colpidium campylum 


200 




24.0° 


No 


Pitts (1932) 




112. 5 




19.8° 


Yes 


Hall (i9j8) 


Colpidium colpoda 


200 




17.0° 


No 


Wachendorff (1912) 




200 






No 


Peters (1929) 


Colpoda sp. 


600-1,200 




19.7° 


No 


Adolph (1929) 


Glaucoma piriformis 




35 


22.0° 


Yes 


M. LwofF (1934) 


Blepharisma undulans 




(0.5)* 


20.8° 


No 


Emerson (1929) 


Spirostomum ambiguum 


2,590 




25.0° 


No 


Specht (1935) 


Strigomonas oncopelti 


0.4 


62 


28.0° 


Yes 


A. Lwoff (1933) 


Strigomonas fasciculata 


0.4 


5? 


28.0° 


Yes 


A. Lwoff (19JJ) 


Leptomonas ctenocephalus 


0.3 


40 


28.0° 


Yes 


A. Lwoff (i93i) 


Trypanosoma equiperdum 


0.05 




37 -0° 


Yes 


Von Fenyvessy and 
Reiner (1928) 


Chilomonas Paramecium 


17-26 




25.0° 


Yes 


Mast, Pace, and Mast 
(1936, 1937) 


Astasia sp. 


2,400 






Yes 


Jay (1938) 


Khaw}{inea halli 


2,050 






Yes 


Jay (1938) 


Actinosphaerium eichhornii 


1,100 




20.0° 


No 


Howland and Bern- 
stein (193 1) 


Amoeba proteus 




(0.2)* 


20.0° 


No 


Emerson (1929) 



Not Q02 but mm^ O2 per hour per mm^ 



tent, and temperature. Since O, consumption of the yellow pigment of 
respiration (see below) varies with Oo tension, it would not be sur- 
prising to find a similar relationship in organisms with this mechanism. 
However, this is not always the case (Schlayer, 1936). Clarification of 



RESPIRATORY METABOLISM 363 

the relationship between O. tension and O. consumption in Protozoa 
will apparently require much more data than is now available. A dis- 
cussion of the theoretical relationship between oxygen tension and oxygen 
consumption is given by Marsh (1935). 

For Protozoa, the available evidence indicates that within wide limits 
O2 tension has little or no effect on the rate of O. consumption for 
Faramecmm and Colpoda, and that it does have an effect on Spirostomum. 
Lund (1918a) found that the rate of O^ consumption for Paramecium 
was independent of O, tension between 0.04 cc. and 2.2 cc. O2 per 
137 cc. — a 55-fold range. This was determined by placing thick sus- 
pensions of Paramecium in stoppered bottles and measuring the dis- 
solved O2 content of the water by the Winkler method, until the animals 
died. Lund's conclusion was confirmed by Amberson (1928), who 
placed the organisms in a closed vessel, in contact with an atmosphere 
of known O, content. By gas analyses he demonstrated a uniform rate 
of Oo consumption, with Oo partial pressures which varied from 
50 to 220 mm. Hg, and only a slight decrease (about 20 percent) at 
pressures as low as 11 mm. Hg. Adolph (1929) found that the O2 
consumption of Colpoda did not vary significantly with Oo tension 
between 155 and 750 mm. Hg. In a single experiment at 4-8 mm. Hg, 
O2 consumption decreased to 31 percent of its previous value. However, 
Adolph did find that low Oo tension (40 mm.) was correlated with 
smaller size of the progeny of cultures. Specht (1935) measured the 
respiration of Spirostomum in pure oxygen, in air, and in 0.5 percent 
O2 in N2. He found that O2 consumption in these gases was in the 
ratio of 151 to 100 to 71, and that COo production was in the ratio of 
175 to 100 to 70. 

When considering the effect of low Oo tensions on Oo consumption 
for any of the larger Protozoa, one should consider the Oo tension at 
various points within the organism as well as at the surface. This can 
be calculated by the diffusion equations of Harvey (1928) and others, 
on the assumption that the rates of cyclosis and water exchange are 
low. The O2 tension at the center of an ellipsoid which is consuming 
O2 uniformly throughout its substance, will be zero when the shortest 
radius 

5Dr 



364 RESPIRATORY METABOLISM 

where D is the diffusion coefficient of O2, c is the O., tension at the 
surface, and A is its rate of O., consumption. In the case of a cyhnder 
(e.g., Spirostomum) the factor 5 should be 4. For Colpoda, Adolph 
(1929) calculated the value of "'<«" to be l48 \x at atmospheric O2 
tension, and 72 \\ at 40 mm. Hg partial pressure of O.,. In large ciliates 
this factor might be important at low Oo tensions, even with a rather 
low rate of O, consumption. 

3. EFFECT OF CO, TENSION ON O, CONSUMPTION 

Root (1930) showed that when CO, tension was raised from 1 mm. 
Hg to 15-20 mm. Hg, the respiration of Paramecium increased slightly 
(less than 15 percent), and it was believed that this increase might be 
caused by increased activity of the organisms. As the CO, tension was 
increased above 60 mm. Hg, O, consumption decreased continuously 
to about 40-60 percent of the control when CO, tension reached 220- 
360 mm. Hg. Similar experiments on fertilized Arbacia eggs did not 
show increase at low concentrations, and all CO, tensions greater than 
30 mm. Hg produced a decrease to less than 40 percent of normal. 
Paramecit/m apparently was much more resistant to increase of CO, 
tension than Arbacia. For both Paramecium and Arbacia only slight 
effects were obtained with HCl, at pH values comparable to those 
present during the CO2 experiment (4.5 to 7.5 for Paramecium). 

From the information available, it is not possible to determine the 
mechanism of action of CO, on protozoan respiration, and results with 
other organisms are few and variable. Apparently CO, is not involved 
as an inhibitor or accelerator of any of the known mechanisms of 
respiration (to be discussed below), and at present we can only say 
that the effect on respiration seems to be indirect, and that the results 
are not due to pH changes in the external fluid. However, internal pH 
changes, as suggested by Root (1930), might account for the effect. 
This is an explanation comparable to that given by Jahn (1936) for 
the effect of the lack of CO., on growth of Chilomonas and bacteria. 
It is well established that certain bacteria will not grow in the absence 
of COg. Jahn (1936) studied the effect of COa-free media on growth 
of Chilomonas and Colpidium and found a distinct inhibition with 
Chilo7nonas and none with Colpidium. The explanation was offered 
that the inhibition of growth might be caused by inadequate intracellular 
buffering in those species which were affected by lack of CO,, and that 



RESPIRATORY METABOLISM 365 

organisms whose normal environment is high in CO. might depend 
more on CO, buffering than those the normal environment of which 
is low in COo. (For possible application of this idea to culture of in- 
testinal forms, see Jahn, 1934). 

4. THE EFFECT OF THE PHYSIOLOGICAL STATE ON Og CONSUMPTION 

It seems as if the effect of various factors which influence the physio- 
logical state of an organism may be reflected in measurements of O^ 
consumption. Factors which have been investigated for the Protozoa are 
starvation, age of the culture, and conjugation. 

Lund (1918c) starved Paramecium in tap water and noted an ap- 
preciable decrease in respiration during the first twenty hours. This was 
simultaneous with the disappearance of deutoplasmic food reserves from 
the protoplasm. Upon feeding starved animals with boiled yeast sus- 
pensions, the rate of oxygen consumption could be increased two to 
three times. This increase was independent of cell division. Leichsenring 
( 1925 ) demonstrated a decrease of 23 percent after twenty-four hours of 
starvation and 29 percent after seventy-two hours. 

The effect of the age of the culture on O2 consumption was first 
studied by Wachendorff ( 1912) , who found that for Colpidium colpoda 
the O, consumed per organism diminished from 191 mm^ per hour 
the first day to 151 mm' the tenth day, and to 59 mm' on the thirtieth 
day. Reidmuller (1936) reported a higher rate of O, consumption for 
young cultures of Trichomonas foetus than for older cultures. Twenty- 
four to forty-eight-hour-old cultures consumed 4.3 mm'' O, per mm'' of 
organisms, sixty-hour cultures, 2.8 mm,' and three-day-old cultures only 
0.81 mm' O,. Andrews and von Brand (1938) reported a decrease 
in sugar consumption per organism for this species, with increasing age 
of the culture, and their data indicate that the differences observed by 
Reidmuller were real, in spite of objections to the method used be- 
cause of the possibility of hydrogen or methane evolution. 

Zweibaum (1921) studied the rate of Oo consumption of Paramecium 
caudatum in relation to conjugation. He found that the rate just before 
conjugation was about 0.73 mm' O2 per thousand organisms per hour. 
During conjugation this rate rose to 3.4 mm'/l,000/hour, and immedi- 
ately after conjugation decreased to about 0.73. During the first eight 
or nine days following conjugation, the rate rose slowly to 2.0 
mm'/l,000/hour, and remained at this value from four to five months. 



366 RESPIRATORY METABOLISM 

5. THE EFFECT OF TEMPERATURE ON O2 CONSUMPTION 

Data concerning the effect of temperature on O^ consumption are not 
numerous, and in most cases are incomplete. Barratt (1905) determined 
the CO2 production of Paramecium at various temperatures and found 
that the rate at 27°-30° C. was more than twice that at 15° C. Wachen- 
dorff (1912) found that C. colpoda respired about four times as fast at 
17° as at 7° C. Leichsenring (1925) demonstrated that Varamecium, 
when transferred from a temperature of 20° C. to one of 35° C, showed 
a respiratory increase of 35 percent, and that when transferred to a 
temperature of 15°, 10°, 5°, 0° C. respiration was decreased 30 per- 
cent, 34 percent, 50 percent, and 58 percent respectively. These effects 
were not completely reversible. The data of Kalmus (1928b) showed 
a Q^„ value (temperature coefficient) of 1.5 for Paramecium respira- 
tion between 23° and 32° C. A. Lwoff (1933) found a Q^^ value of 2.1 
between 13° and 23° C, and about 1.5 between 23° and 32° C. The 
temperature characteristic (p value) was 9,830 calories for the range 13° 
to 34° C. Lwoff also calculated a |j value of 21,350 for the synthesis 
of respiratory enzyme (oxidase, see below) by the organism between 
18° and 31.5° C, and a value of — 52,000 between 31.5° and 
34.0° C. 

6. THE EFFECT OF ANESTHETICS AND POISONS ON O, CONSUMPTION 

The effect of various toxic agents (e.g., KCN, CO, N3H, arsenite, 
urethanes, and so forth) which are supposed to exert a specific effect 
on the normal functioning of certain respiratory enzymes, will be dis- 
cussed in connection with the mechanism of respiration. The effect of 
other anesthetics on respiration has not been extensively studied. Leich- 
senring (1925) found that ethylene and nitrous oxide had no effect on 
the respiration of Paramecium, and that ether and chloroform produced 
decreases of as much as 25 percent (after two and one hours respec- 
tively). The effect was reversible with ether, but not with chloroform 
if the exposure was more than one-half hour. Colpoda was more sensitive 
to these substances than Paramecium. Von Fenyvessy and Reiner (1928) 
found no decrease in respiration of Trypanosoma equiperdum when one- 
percent Germanin was added. This was surprising, in view of the fact 
that Germanin is very toxic for trypanosomes in vivo. 



RESPIRATORY METABOLISM 367 

7. THE EFFECT OF NUTRITIVE SUBSTANCES AND OTHER MATERIALS 

The effect of various nutritive substances on Protozoa has been demon- 
strated by a number of investigators, but the criteria used are usually 
growth or the accumulation of food reserves, and not respiration. In 
some cases it is possible to determine that the substance is oxidized 
directly (e.g., glucose), or that it contributes toward the synthesis of 
the respiratory enzyme (A. Lwoff, 1933). These data will be dis- 
cussed below. The effect of various substances on the respiration of 
Paramecium was studied by Leichsenring (1925), who found that cap- 
rine, glutamic acid, peptone, and aminoids increased respiration 12-18 
percent; that glycocoU and succinic acid increased respiration 8-9 per- 
cent; and that tyrosine and cystine produced little effect. Lactose gave 
an increase of 16 percent, and other sugars and polysaccharides gave in- 
creases of 3-10 percent. Thyroxin gave an increase of 13 percent. No 
explanation was made of the mechanism of these effects. 

Mast, Pace, and Mast (1936) found that Chilomonas grew well, 
formed considerable starch but little fat, and consumed 0.17 mm^ of O2 
per 10,000 organisms per hour, in a solution of MgSO^, NH^Cl, 
K2HPO4, Na-acetate, and silicon. When sulphur was omitted, the starch 
remained constant, fat accumulated, Oo consumption decreased, and the 
animals finally died. When acetate was omitted, the organisms decreased 
in size, starch and fat decreased, and O, consumption decreased to 0.07 
mmY10,000/hour. When both sulphur and acetate were omitted, starch 
decreased to zero, fat accumulated, Oo consumption decreased, and the 
organisms died. These authors conclude that starch is normally changed 
to fat, that sulphur induces oxidation of fat, thereby increasing respira- 
tion and preventing accumulation of fat, and that fat oxidation is 
probably associated with a cystine-cysteine mechanism (see glutathione, 
below). Mast and Pace (1937) reported a 25 percent increase in res- 
piration of Chilomonas when Na^SiOg was added to inorganic media. 
This was supposedly caused by the catalytic action of Si on organic 
syntheses. 

8. EVOLUTION OF GASES OTHER THAN COg 

The possibility that Protozoa evolve gases other than CO2 was first 
shown by Cook (1932) for the flagellates of termites [Termopsis neva- 
densis). A gas which was not absorbable by hydroxide was evolved by 



368 RESPIRATORY METABOLISM 

normal termites, under anaerobic conditions. However, this gas was not 
evolved if the intestinal flagellates had been removed by oxygenation, 
and it was suggested that it might have been evolved by the flagellates. 
The evidence is incomplete, however, in that oxygenation probably also 
changed the bacterial flora. Witte (1933) observed the production of 
gas bubbles by Trichomonas foetus. This was confirmed by Andrews and 
von Brand (1938), who found that this gas was not absorbable by 
alkali and that when mixed with O2 it burned with indications of ex- 
plosiveness. Final identification was not made. The formation of gas 
vacuoles has been reported for several organisms, but in most cases there 
is little evidence regarding the identity of the gas. Bles (1929) believed 
that the gas vacuoles of Arcella contained Oo. 

The chlorophyll-bearing flagellates, of course, might give off Og in 
the presence of strong light, because of photosynthesis, and this might 
also be true of the ciliates which harbor zoochlorellae. There is con- 
siderable indirect evidence that this is true, but no direct measurements 
are available. 

Investigations Which Concern the Source of Energy 

Whenever an oxidizable material is subjected to complete combustion, 
the ratio of COg given off to Oo consumed will vary with the type of 
material. This ratio (COg/Oo) is called the respiratory quotient (R.Q.). 
Carbohydrates (relatively rich in oxygen) have an R.Q. of 1.0; fats 
(relatively poor in oxygen) have an R.Q. of about 0.71; and proteins 
have an R.Q. of 0.83 if the nitrogen is eliminated as urea, and 0.93 if it 
is eliminated as ammonia. By measurements of the respiratory quotient 
we may obtain an index of the type of material which is being oxidized 
by the organism, at least as to whether it is predominantly carbohydrate 
or predominantly fat. If the excreted nitrogenous material can be identi- 
fied and measured, the amount of protein and consequently the amounts 
of carbohydrates and fats consumed can be calculated. Under these con- 
ditions the measurement of R.Q. becomes more significant. 

These interpretations are based on the assumptions that complete 
oxidation of metabolic substrates is the sole cause of gaseous exchange, 
and that gases other than Oo and CO, are not involved. If these assump- 
tions are unsound, then any interpretation of the R.Q. is necessarily 
more difficult. If carbohydrate is being converted into fat within the 



RESPIRATORY METABOLISM 369 

organism for purposes of storage, the R.Q. may reach a value of 1.4. 
If fat or protein is being converted to carbohydrate, there will be a cor- 
responding tendency for the R.Q. to be lowered. A high value for the 
R.Q. can also arise whenever CO, is removed from a compound with- 
out the consumption of O^, or whenever an oxygen debt accumulates 
(during heavy exercise). Whenever an oxygen debt is being removed, 
the R.Q. may fall to extremely low values (during rest after exercise). 
Unusual values of R.Q. may be obtained if substrates other than carbo- 
hydrates, fats, or proteins are being consumed. In the case of very rapid 
protozoan growth on substrates of organic acids, the R.Q. should vary 
with the oxygen content of the molecule being oxidized (acetic acid, 
1.0; proprionic acid, 0.85; butyric acid, 0.80). 

The respiratory quotient of an organism may be calculated with almost 
any of the manometric methods described above, if separate measure- 
ments are made with and without a COa-absorbing alkali in the respira- 
tory chamber. In this case, one set of readings (with KOH) will give a 
measure of the O2 consumed, and the other set (without KOH) will 
be an Index of the difference between CO2 given off and O2 consumed. 
From this the R.Q. may be calculated, provided no NHg is evolved and 
no CO2 is retained in the immersion fluid. If these complications arise, 
suitable modifications may be introduced. The use of somewhat more 
complicated manometer chambers allows measurements of simultaneous 
O2 consumption and CO2 production to be made on the same material 
(Dixon, 1934). For the Protozoa this method seems preferable because, 
in addition to its usual advantages, it prevents the results from being 
affected by the possible secretion of ammonia (Specht, 1935) and 
other bases (e.g., sodium carbonate from oxidation of Na-acetate, Jahn, 
1935a). However, even this method does not correct for the possible 
evolution of hydrogen or methane (cf. Trichomonas foetus, Andrews 
and von Brand, 1938). 

For the ciliates, several measurements of R.Q. have been made. Wa- 
chendorff (1912) reported R.Q. values of about 0.3 for Colpidium, but 
in view of later developments it seems as if this material should be re- 
examined with more modern methods. Emerson (1929) studied the 
respiration of Blepharisma undulans and found an R.Q. slightly less 
than 1.0. Daniel (1931) obtained an R.Q. of 0.84 for Balantidium colt, 
but the possible effects of bacteria were not well controlled. Amberson 



370 RESPIRATORY METABOLISM 

(1928) reported an R.Q. of 0.69 for Paramecium, and Root (1930) 
in a number of experiments obtained an R.Q. value of 0.62. Root also 
found that the R.Q. varied somewhat irregularly with changes in COg 
tension. However, there was a definite trend toward high R.Q. values 
in media of high COg tension, and the average R.Q. at 238-423 mm. 
Hg. was 1.43. This apparently was caused by a decrease in O^ consump- 
tion (see above) without a corresponding decrease in CO2 production, 
thereby giving a high R.Q. According to Root, "It is possible that the 
suppression of oxidations under these conditions results in the produc- 
tion of acid metabolites which drive out carbon dioxide from the bi- 
carbonate contained in the cells and in the surrounding medium." Similar 
experiments on Arbacia eggs did not show an increase in R.Q., and it 
was assumed either that acid substances were not produced or that they 
were rapidly converted into a non-acid form and did not accumulate 
in appreciable amounts. Apparently CO, tension is a factor which should 
be considered when making measurements of R.Q. However, if ex- 
periments are conducted with standard manometric techniques, this factor 
is probably not important. 

Specht (1935) measured the R.Q. of Spirostomum in manometers, 
both with and without the presence of acid in a side arm of the manom- 
eter flask. He found an R.Q. of 0.24 without the acid and 0.84 when 
acid was present. This discrepancy was explained as being caused by 
the elimination of NH3 by the organisms, and the value of 0.84 is 
therefore accepted as more nearly correct. However, it was also demon- 
strated that the R.Q. was 0.98 in an atmosphere of O,, and that this 
value was not affected by the presence or absence of acid. Apparently 
NH3 is not produced at high O^ tension. These experiments indicate 
very clearly that ammonia secretion is a possible source of error in 
measurements of protozoan respiratory quotients, and therefore one 
should be suspicious of the validity of low R.Q. values unless adequate 
precautions have been taken against the ammonia error. 

For the free-living flagellates, very low values of R.Q. have been 
reported. Jay (1938) found R.Q. values of 0.34 and 0.56 for Astasia 
and Khatvkinea, respectively. Mast, Pace, and Mast (1936) reported 
R.Q. values of 0.28 to 0.37 for Chilomonas , and 0.72 for Paramecium 
fjjulti)7iicronucleatum under similar conditions. The possible explana- 
tions mentioned by Jay for the low R.Q. value are conversion of pro- 



RESPIRATORY METABOLISM 371 

tein to carbohydrate, or incomplete oxidation of carbohydrate. The ex- 
planation of Mast et al. is that carbohydrate was being synthesized from 
carbon dioxide. This explanation is based on previous nutritional studies, 
but these are open to question (review. Hall, 1939). One obvious possi- 
bility is that COo may be retained in the immersion fluid, but Mast 
and his coworkers obtained only a slightly higher R.Q. value when the 
bound COo was liberated by acid (single experiment only). In these 
cases the explanations offered must be considered as only tentative, until 
the possibilities of NH3 production and COo retention are positively 
eliminated. Mast and his coworkers also reported for Chilomonas that 
under certain conditions starch was converted to fat, and that fat oxida- 
tion could be decreased by depriving the organism of sulphur. However, 
values obtained for the R.Q. were variable and showed no definite cor- 
relation with these conditions. 

Values of the R.Q. reported for members of the family Trypano- 
somidae are within the normal range. Soule (1925) obtained an R.Q. 
of 0.84-0.91 for Leishmania tropica and 0.74-0.89 for Trypanosama 
lewisi in blood agar medium. When glucose was present, the R.Q. rose 
to 0.95 for L. tropica and 0.94 for T. letvisi. Novy (1932) reported res- 
piratory quotients of 0.93 to 1.0 for T. lewisi, L. tropica, L. donovani, L. 
infantum, Strigonionas oncopelti, S. culicidarum, S. culicidarum var. 
anophelis, S. lygaeorium, S. media, S. muscidarum, and S. parva, when 
grown on glucose-blood agar. When grown on glycerol-blood agar or 
plain blood agar, the R.Q. was about 0.8 to 0.87 for the four species of 
Leishmania. Von Fenyvessy and Reiner (1924) found an R.Q. of 0.60 
for Trypanosoma equiperdum in diluted blood. A. Lwoff (1933) ob- 
tained R.Q. values of 1.0 for Strigomonas oncopelti and S. jasciculata, 
and a value of 0.88 for Leptomonas ctenocephali. 

Apparently the only R.Q. measurement on a rhizopod is that of 
Emerson (1929) on Amoeba proteus, which gave a value slightly less 
than 1.0. 

Another method, in addition to that of the respiratory quotient, which 
might be used as an index of the source of energy in an organism is 
the calorific quotient. Since the ratio of heat produced to oxygen con- 
sumed differs with carbohydrate, fat, and protein (3.5, 3.3, and 3.2, 
respectively), it is possible to measure heat production and Oo con- 
sumption and to use this ratio as an index of the substrate being utilized. 



372 RESPIRATORY METABOLISM 

However, because of the small differences in the ratios and because of 
the complications of the technique of heat measurement, the calorific 
quotient has not been found to be very useful in determining the energy 
source for Metazoa (Needham, 1931), and apparently has not been 
tried for Protozoa. 

Investigations Which Concern the Mechanism of Respiration 

1. general theory 

For a general consideration of the mechanism of respiration, the 
reader is referred to the monographs of Meldrum (1934) and Holmes 
(1937), to standard textbooks of general physiology, to several excel- 
lent discussions in recent volumes of the Annual Revietv of Biochemistry, 
and to the forthcoming volume of the Cold Spring Harbor Symposia in 
Quantitative Biology (Vol. VII). The present discussion of the mecha- 
nism of respiration will include only those portions of a bare outline 
which are necessary for an understanding of the data and interpretations 
which are to follow. 

The first step in oxidation of a substrate is the removal of hydrogen 
from the substrate molecule, and the addition of this hydrogen to any 
other molecule which will serve as a hydrogen acceptor. After de- 
hydrogenation is accomplished, the resulting molecule is supposed to be 
very unstable and easily undergoes oxidation by molecular oxygen, to 
form CO2 and water. The enzymes necessary for these final stages in 
respiration are not well known, but the enzymes and respiratory pig- 
ments responsible for dehydrogenation and the subsequent transfer of 
the hydrogen to Oo with formation of water are listed below. (Any 
distinction between respiratory enzymes and pigments is purely arbi- 
trary. ) 

( 1 ) Dehydrogenases are enzymes which bring about activation of the 
substrate, so that it may be oxidized by oxygen or intermediate hydrogen 
acceptors such as cytochrome. These enzymes are highly specific, in that 
they react with only one or a few substrates. Dehydrogenases are divided 
into two groups : anaerobic dehydrogenases, which cannot reduce molecu- 
lar O2 in the presence of their substrates, and aerobic dehydrogenases 
which can do so. Cytochrome and cytochrome oxidase are important 
factors in the completion of oxidation by anaerobic dehydrogenases. 



RESPIRATORY METABOLISM 373 

(2) Cytochrome is a group of pigments or enzymes, which in the 
living cell are oxidized under aerobic, and reduced under anaerobic 
conditions, but which cannot be oxidized directly by molecular O,. These 
serve as hydrogen acceptors for anaerobic dehydrogenase systems. 

(3) Oxidase. — The term oxidase includes all enzymes which are 
capable of performing oxidations in the presence of molecular oxygen. 
To this group belongs the respiratory enzyme of Warburg, which is 
perhaps identical with the oxidase of cytochrome, which in turn is 
also referred to as indophenol oxidase because of one method of detect- 
ing its activity. This enzyme brings about the oxidation of reduced 
cytochrome by molecular oxygen, and water is supposed to be oxidized 
to hydrogen peroxide during the process. Aerobic dehydrogenases are 
sometimes classified as oxidases. 

(4) Catalase is an enzyme present in aerobic organisms and usually 
absent in anaerobes. This enzyme converts hydrogen peroxide to water 
and molecular oxygen, and its place in the respiratory chain is given 
below. 

(5) Peroxidases are enzymes which in the presence of an oxidizable 
substrate convert hydrogen peroxide to water and activated oxygen, 
thereby causing oxidation of the substrate. The exact role of peroxidases 
in cellular respiration is not understood. The peroxidases are iron com- 
pounds, and other iron compounds, such as cytochrome and methemo- 
globin, exhibit some peroxidase-like activity. 

(6) Yelloiv respiratory pigment, or enzyme, is a flavo-protein capable 
of reversible oxidation and reduction whicii may be reduced in a re- 
action involving oxidation of substrate (through the intermediary action 
of a co-enzyme) and which can then be reoxidized in the presence of 
molecular Oo (with formation of HoOo) or other hydrogen acceptors. 

(7) Glutathione is an amino-acid complex capable of reversible oxi- 
dation and reduction, and which may act as a hydrogen acceptor through 
the reduction of an -S-S- group to two -SH groups (cysteine to 2 
cysteine) which are auto-oxidizable in the presence of molecular Og. 

The details of how these substances function in the living cell are 
subject to considerable controversy, but for our present purpose we may 
regard the general outline for the first four items listed above as follows: 



(1) substrate -|- 2 oxidized cytochrome ^ 



dehydrogenase 

oxidized substrate -j- 2 reduced cytochrome 



374 RESPIRATORY METABOLISM 

( 2 ) 2 reduced cytochrome -f- O, ==^ 2 oxidized cytochrome -j- HgOg 

oxidase 

(3) HA^=^ H,0 + l/2 0, 

catalase 

The substrate may be activated by "anaerobic" dehydrogenase, and 
it is then oxidized by cytochrome, the cytochrome itself being reduced 
in the process (equation 1). Cytochrome is, in turn, oxidized by an 
oxidase system which may be identical with Warburg's respiratory en- 
zyme (equation 2). During this process HoOo is formed and is then 
broken down to water and molecular O2 by catalase (equation 3). 

The oxidase and catalase systems are inhibited by the presence of 
HCN and HoS, and the oxidase system is also inhibited by CO. In the 
presence of any of these reagents, reaction ( 1 ) can proceed but not 
reactions ( 2 ) or ( 3 ) . Therefore all of the cytochrome becomes reduced, 
and respiration by means of this mechanism is stopped. The dehydrogen- 
ase systems are inhibited by narcotics (e.g., the urethanes), by warm- 
ing and cooling, and these agents leave all of the cytochrome in the 
oxidized state. These two general methods of treatment, therefore, may 
be used as tools in studying the above respiratory mechanisms. There are 
also aerobic dehydrogenases which, in addition to activating the sub- 
strate, can react directly with molecular oxygen without the mediation 
of cytochrome and oxidase. Respiration which is brought about by this 
type of system is not supposed to be affected by HCN. 

It is possible to demonstrate that in some systems certain reversible 
oxidation-reduction indicators (e.g., methylene blue) can replace the 
cytochrome-cytochrome oxidase system, and that in this capacity the 
action of these indicators may or may not be affected by HCN and CO 
(e.g., grasshopper embryos, Bodine and Boell, 1937; Escherichia coli, 
Broh-Kahn and Mirsky, 1938). If HCN and methylene blue are added 
to such a respiratory system and inhibition does not occur, the ensuing 
reactions might be visualized as follows: 

(4) substrate -j- methylene blue ^ — oxidized substrate 

dehydrogenase 

-j- leuco-methylene blue 

( 5 ) leuco-methylene blue -j- oxygen ^ methylene blue -{- H^Og 

Since catalase is inactivated by HCN, the hydrogen peroxide presumably 



RESPIRATORY METABOLISM 375 

accumulates and in E. coU cultures can be measured experimentally. In 
anaerobes which normally do not possess catalase, this mechanism might 
explain the bacteriostatic effect of oxidation-reduction indicators. 

It is also known that there are certain pigments of bacteria and yeasts 
(e.g., yellow enzyme, or pyocyanine) which are capable of bringing 
about a similar result, and other naturally occurring oxidation-reduction 
indicators have been described (echinochrome, hermidin, and pigments 
from B. violaceus and Chromodoy'is zebra) which apparently might func- 
tion in a similar fashion. The reactions involving yellow pigment and 
its coenzyme may be indicated as follows: 

(6) substrate -f- coenzyme - oxidized substrate -\~ reduced 

dehydrogenase 

* coenzyme 

(7) reduced coenzyme -[- yellow pigment ;:± coenzyme -j- 

leuco-yellow pigment 

(8) leuco-yellow pigment -|- oxygen ^ yellow pigment -j- H2O2 

(9) H,0,^=~ H,0 + l/2 0, 

catalase 
In this case only the action of catalase is prevented by HCN, and there- 
fore H2O2 accumulates. In the absence of Oo the leuco-yellow enzyme 
may be oxidized by other substances (e.g., by methylene blue). The 
yellow enzyme has been found to be a combination of protein and 
vitamin G, and it is believed that while this sort of system is present 
in aerobic organisms, it assumes its greatest importance in anaerobic 
species. In anaerobic organisms (yellow enzyme can be prepared from 
bottom beer yeast or lactic acid bacilli) we have, then, a respiratory 
system which is quite independent of cytochrome and Warburg's oxidase, 
and which therefore is insensitive to HCN and CO. Perhaps when we 
say that the respiration of a given species is cyanide insensitive, we may 
be inferring that that species has a respiratory system more suited to 
anaerobic conditions (temporary or otherwise). Under anaerobic condi- 
tions the leuco-yellow pigment is probably oxidized by substances other 
than molecular oxygen, and HgOg is not formed. The known respiratory 
enzymes of bacteria are summarized by Frei (1935) and Stephenson 
(1939). 

The relationships between the various respiratory enzyme systems 



376 RESPIRATORY METABOLISM 

are not so well known nor so clear-cut as, for the sake of clarity and 
brevity, they have been made to appear in the above outline. Whenever 
we conclude, on the basis of the action of certain reagents on respiration, 
that one respiratory mechanism is very important and that another is 
not, we should do so only with certain mental reservations, and the 
conclusions should not be considered final, but merely indicative. 

2. EXPERIMENTS WHICH CONCERN THE CYTOCHROME-CYTOCHROME 
OXIDASE SYSTEM OF HYDROGEN ACCEPTORS 

We have, through the action of HCN and CO on respiration, a tool 
for determining how much of the respiratory activity of a given organism 
is carried on by means of the cytochrome-respiratory enzyme system and 
how much is not. Respiration which is not cyanide and CO sensitive 
may be due to aerobic dehydrogenases, or to anaerobic dehydrogenases 
plus an enzyme of the yellow-pigment type or perhaps to the action 
of peroxidases. Such analyses have been made for several types of bio- 
logical material. It has been determined that respiration of some cells 
is extremely sensitive to HCN (e.g., yeast, B. col/, most bacteria, and 
mammalian tissues), while that of others is quite resistant [Chlorella, 
Paramecium, Sarcina, Pneumococcus, B. acidophilus. Streptococcus, 
Staphylococcus); also, the same organism may differ in sensitivity at 
different periods during its life history (grasshopper eggs, Robbie, Boell, 
and Bodine, 1938). One technical precaution which should be ob- 
served in cyanide experiments is the use of a KOH-KCN absorbing 
fluid for COo (van Heyningen, 1935). By the selection of the proper 
KOH-KCN mixture, the osmotic transfer of KCN through the air from 
the experimental material to the KOH solution can be prevented. This 
is apparently one possible source of error in all work on the effect of 
cyanide on protozoan respiration — that none of the authors has used 
balanced KOH-KCN solutions. 

Among the Protozoa the effect of cyanide has been studied on several 
ciliates and flagellates. It is quite well established that the respiratory 
mechanism of Para?necium is insensitive to cyanide (Lund, 1918b; Shoup 
and Boykin, 1931; Gerard and Hyman, 1931), and the work of Shoup 
and Boykin (1931) shows that the addition of iron salts does not 
increase respiration and that very little or no iron is present in Para- 
mecium. These results may be interpreted to mean that the cytochrome- 



RESPIRATORY METABOLISM 377 

respiratory enzyme system plays no part in the respiration of Paramecium. 
Peters (1929) obtained no inhibition with M/500 KCN on Colpidium 
colpoda. The data of Pitts (1932) for C. campylum shows that less than 
20 percent of the respiration is cyanide sensitive and that this depression is 
only temporary, and that while still in cyanide the respiration may rise 
to a rate which is as much as 25 percent above normal and drop again 
to 80 percent of the normal rate. M. Lwoif (1934) found that the 
respiration of Glaucoma prriformh in peptone solution was depressed as 
much as 80 percent during the first half hour, but that by the third 
hour the rate had returned to normal (M/1,000 KCN), or almost 
normal (15 percent below in M/450 KCN), and then decreased. In a 
weaker KCN solution (M/4,500) this latter decrease did not occur. 
The organisms were able to live twenty- four hours in M/450 KCN 
and eight days in M/1,000, but they did not divide. In M/2,000 and 
M/5,000 KCN multiplication of the organisms occurred slowly. In 
glucose-Ringer solution, M/450 KCN did not inhibit, but produced an 
acceleration of as much as 36 percent during the first half hour. The 
conclusion to be drawn from these data is that the ciliates, as far as we 
know, are relatively insensitive to the action of cyanide, and we might 
consider the temporary inhibitions produced in some cases as secondary 
effects rather than direct effects upon the respiratory mechanism (cf. 
another explanation mentioned below). It would be interesting to rein- 
vestigate the effect of KCN on some of these ciliates by the use of 
balanced KCN-KOH solutions. It seems possible that the data, especially 
in the case of the temporary effects, may be complicated by the loss of 
KCN from the experimental solution to the KOH solution. 

Among some of the flagellates, however, there seems to be quite a 
different respiratory mechanism. A. Lwoff (1933) found that M/3,000 
KCN inhibited respiration of Strigomonas oncopelti 90 percent, of 
5". jasciculata 83 percent, and of Leptofnonas ctenocephali 95 percent. 
With M/1,000 KCN, both species of Strigomonas were inhibited 90 
percent, and L, ctenocephali 95 percent. The latter species was extremely 
sensitive and was inhibited 92 percent by M/20,000. Growth was also 
decreased in those concentrations which inhibited respiration, and the 
organisms were killed only by much greater concentrations. M. Lwoff 
(1934) reported an inhibition of 90 percent for Polytoma uvella, and 
Jay (1938) an inhibition of 60-65 percent for Khawkinea and Astasia 



378 RESPIRATORY METABOLISM 

at a concentration of M/lOO KCN. Von Fenyvessy and Reiner (1928), 
however, reported no effect with 0.1 percent KCN (M/65) on either 
oxygen consumption or acid production of Trypanosoma equiperdum in 
glucose-bicarbonate-Ringer solution. 

These results demonstrate that the respiratory mechanisms of various 
Protozoa are probably not the same. The respiratory mechanisms of some 
Protozoa seem to resemble those of Chlorella and Sarcina, while those 
of other species resemble the mechanisms of yeast and mammalian and 
other tissues. This question is one which should be studied carefully 
in a wide variety of organisms, and with a wide concentration range 
of cyanide solutions. The taxonomic position of the Protozoa should 
make such an investigation doubly interesting. It would also be of 
interest to know if the cyanide insensitivity of Paramecium is still main- 
tained in the presence of glucose and other substances, or if an apparent 
change in the respiratory mechanism is brought about by the presence 
of glucose. Emerson (1929) found that the respiration of Chlorella was 
cyanide sensitive only in the presence of glucose; Gerard (1931) found 
that glucose had no effect on the cyanide sensitivity of Sarcina, but 
M. Lwoff (1934) found that the respiration of Glaucoma was accelerated 
by HCN when glucose was absent. These divergent results should have 
a final explanation in terms of the respiratory or other metabolic mecha- 
nisms involved. 

An alternative theory to the supposed coexistence of cyanide sensitive 
and insensitive fractions in the normal cell is that all normal respiration 
is CN sensitive, and that in the presence of CN an entirely new 
respiratory mechanism is called into existence. This interpretation would 
indicate that among many bacteria, algae, and ciliates (but not among 
certain flagellates) there is a greater adaptability of the respiratory 
mechanism than among the more specialized cells of the Metazoa. Such 
generalizations are probably premature, but it does seem possible that 
aerobic protozoa which can live anaerobically for considerable periods 
of time might have a dual respiratory mechanism. 

It has been demonstrated for several biological materials that respira- 
tion sensitive to cyanide is also sensitive to CO (because of CO-inhibi- 
tion of cytochrome oxidase), and that respiration insensitive to cyanide 
is not depressed and may even be stimulated by CO (literature cited 
by Bodine and Boell, 1934). A. Lwoff (1933) showed that the KCN 



RESPIRATORY METABOLISM 379 

sensitive fraction (90 percent) of S. fasciculata respiration was also 
sensitive to CO. In an 80/20 mixture of CO/O, inhibition was 61 percent, 
in 95/5 mixtures 85 percent, and in 98/2 mixtures 90 percent. Values 
of K for the Warburg-Negelein equation 

A CO 

where A is O, consumption in the CO/Oo mixture and A,, is O. con- 
sumption in the control, varied from 2.58 in 80 percent CO to 5.3 in 
98 percent CO. M. Lwoff found that carbon monoxide (2-5 percent Oo 
in CO ) produced the same effect on Glaucoma as KCN in both peptone 
and in glucose-Ringer solutions. In peptone there was a marked inhibi- 
tion for the first half hour and then a return to normal or almost normal, 
and in glucose-Ringer there was an increase of 20 percent. Reidmuller 
(1936) reported no appreciable effect of CO on O. consumption for 
Trichomonas joetus in 95/5 mixtures of CO and Oo. The effect of KCN 
was not investigated. The effect of CO on other Protozoa should be in- 
vestigated together with the effect of cyanide. Recently azide (HN3) 
has been found to have an effect on respiration which is similar to but 
not identical with that of HCN and CO (Keilin and Hartree, 1936; and 
others), and it would be interesting to make comparisons of these re- 
agents on protozoan material. 

For the purpose of inhibiting the cytochrome-cytochrome oxidase sys- 
tem, CO is apparently much more specific then HCN or azide. This is 
especially true if inhibition accurs in the dark but not in the presence 
of bright light, because the inactive compound formed by CO and 
cytochrome oxidase is dissociated upon illumination into CO and active 
oxidase. The reversibility upon illumination of CO inhibition has not 
been investigated for Protozoa. 

The distribution of cytochrome among the Protozoa is a relatively 
untouched subject. A. Lwoff (1933) found two absorption bands in 
Strigomonas fasciculata, one at 530, and another rather broad band at 
555 mp. These bands disappeared upon passage of O2 through the solu- 
tion. Upon addition of KCN no other bands became visible, and the 
question arises as to whether the 555 band was the b and c bands of cyto- 
chrome or the b band of the hemochromogen, as has been found for 
various bacteria. Lwoff found the 555 band also in S. oncopelti, Glau- 



380 RESPIRATORY METABOLISM 

coma phijormh, and Euglena gracilis. By treating G. piriformis and 
Polytoma uvella with sodium hydrosulphite and pyridine, he obtained 
the bands of pyridine-hemochromogen. The observations on Glaucojna 
are especially interesting because respiration is KCN and CO insensitive, 
and the explanation of the KCN and CO experiments therefore needs 
further clarification. Perhaps these organisms contain both cytochrome 
and a KCN insensitive system (e.g., yellow pigment) which may func- 
tion interchangeably. This would explain their adaptability to both aerobic 
and anaerobic conditions, the presence of cytochrome and KCN and CO 
insensitivity, and perhaps also the somewhat oscillatory character of 
Colpidium and Glaucoma respiration in the presence of KCN (Pitts, 
1932; M. Lwoff, 1934). This, of course, is pure speculation. However, 
the possibility of any discrepancies in the supposed parallelism between 
CN, CO, and HNo insensitivity and the absence of cytochrome should 
warrant an intensive investigation. Reidmuller (1936) was unable by 
spectroscopic methods to find either cytochrome or hemochromogen in 
Trichomonas foetus, and this result should be expected because of the 
CO-insensitivity of Trichomonas respiration. 

3. EXPERIMENTS WHICH CONCERN OTHER SYSTEMS OF HYDROGEN AC- 
CEPTORS 

If we assume that the Warburg-Keilin system is not present in the 
ciliates, then we must seek another respiratory mechanism. Is this to be 
found in the action of glutathione? According to M. Lwoff (1934), the 
effect of arsenious acid on respiration offers a tool for detecting the action 
of glutathione because it is not supposed to affect the Warburg-Keilin 
respiratory system and because it does combine with -SH groups, thereby 
inhibiting the normal functioning of glutathione. In Glaucoma pirifortnis 
M. Lwoff found that M/1,900 arsenious acid (neutralized sodium ar- 
senite) inhibited 75-80 percent of the respiration and that M/1,150 
inhibited 90 percent. The organisms moved slowly in M/400 to M/2,000 
and remained alive more than thirteen days in M/6,000, but did not 
multiply. The inhibition of respiration was entirely reversible (recovery 
in 1 1/2-2 hours from M/2,000). Monoiodoacetic acid, at least in some 
cases, is supposed to be similar in its action to arsenious acid, that is, it 
combines with -SH. (In other cases its action may be different, e.g., in 
the prevention of lactic-acid production from glycogen in muscle extracts 



RESPIRATORY METABOLISM 381 

in which glutathione is not present. ) Therefore we may use it as an ad- 
ditional indicator in detecting the action of glutathione in respiration. 
M. Lwoff found that monoiodoacetic acid produced 61-82 percent in- 
hibition of respiration of Glaucoma in concentrations of one part in 
121,000 to 77,000, while the cihates appeared normal and moved slowly. 
These results are interpreted to indicate that glutathione seems to be 
quite important in the respiration of Glaucoma. Another interpretation 
which has been used for work on other material (Korr, 1935; Cohen and 
Gerard, 1937) is that arsenites inhibit dehydrogenases (Szent-Gyorgyi 
and Banga, 1933). It is interesting that the accepted dehydrogenase in- 
hibitors (urethanes, see below) do not result in as great an inhibition 
with Glaucoma as arsenites and monoiodoacetic acid. 

Another respiratory mechanism which might exist among the cyanide- 
insensitive Protozoa is the yellow pigment found in yeast and other an- 
aerobic organisms. It seems as if an investigation of the distribution of 
enzyrnes of this type should be made among the Protozoa, especially with 
those species in which respiration proves to be cyanide insensitive. Since 
a large number of Protozoa are presumably facultative anaerobes, it 
might be possible to poison the normal aerobic mechanism and study the 
anaerobic mechanisms under various conditions, as has been done for 
Escherichia coli by Broh-Kahn and Mirsky (1938). 

4. INHIBITION OF THE DEHYDROGENASE SYSTEM 

The dehydrogenases are apparently a part of the respiratory chain in- 
volved in several of the enzyme mechanisms. Therefore one would ex- 
pect any substance which inhibits the dehydrogenases to inhibit 
respiration. Such is the case with the urethanes. M. Lwoif (1934) found 
that in the respiration of Glaucoma one percent methyl urethane pro- 
duced 9 percent inhibition of respiration, 2 percent inhibited 38 percent, 
2.5 percent inhibited 52 percent, and 3.5 percent inhibited 55-63 percent. 
Ethylurethane in concentration of 1.66 percent inhibited 44 percent, and 
2 percent inhibited 57-61 percent. Propylurethane in 0.5 percent solu- 
tion produced an inhibition of 47 percent. The ciliates appeared normal, 
movement was slow, but the effect on respiration was reversible. There- 
fore we may conclude that dehydrogenase systems are probably involved 
in the respiration of Glaucoma. It might be interesting to try the com- 
bined effects of urethane and arsenious acid, in order to obtain evidence 



382 RESPIRATORY METABOLISM 

for or against the idea that both dehydrogenase and glutathione are part 
of the same respiratory chain. 

5. SYNTHESIS OF RESPIRATORY ENZYMES VITAMINS 

After we have determined that a certain organism has a certain type of 
respiratory system, the question arises of how the enzymes are formed. 
Is the organism capable of synthesizing them from relatively simple 
compounds, or must certain prosthetic groups be present in the nutritive 
substrate? For certain flagellates this question has been answered very 
definitely by A. Lwoff ( 1933) . Strigonionas oncopelti, S. jasckulata, and 
Leptomonas ctenocephdi have respiratory systems which are 90 percent 
dependent upon cytochrome (as demonstrated above with KCN and 
CO). It was found that S. oncopelti could live indefinitely in peptone 
solutions without the addition of hematin compounds. For the other 
two flagellates, hematin compounds were found to be necessary. L. 
ctenocephdi would not grow unless rabbit blood (or an equivalent 
amount of hematin) were present in concentrations of one part to 1,200. 
S. jasciculata showed growth in blood dilutions as great as 1/1,000,000, 
and within limits the amount of growth was directly proportional to the 
amount of blood. Hemoglobin disappeared rapidly from the culture 
medium, and it apparently was being used to form more respiratory 
enzyme. When small amounts of blood were added, the Qo^ increased 
linearly for several hours, until apparently all of the hematin was con- 
verted into respiratory enzyme; then the Q02 remained constant. This 
constant level varied with the amount added. It was found that for each 
gamma of blood (between one and 5 gamma) added to 1 mgm. of 
flagellate (dry weight) the Qoo was increased about 3 units. After 8.5 
hours 5 gamma of blood raised the Qo. from 19-5 to 37.0. Blood could 
be replaced with hematin, prohemin, and protoporphyrin, but not by cyto- 
chrome C nor by a wide variety of synthetic hematin and porphyrin com- 
pounds, chlorophyll, peroxidase, or active iron (Lwoif, 1938). Ap- 
parently only the porphyrin compound which contained the vinyl (-CH 
= CHo) radical (protoporphyrin) was effective. Deuteroporphyrin, 
which differs from protoporphyrin in having hydrogen in place of the 
vinyl groups, was not effective. More recent investigations of the chem- 
ical structure of cytochrome indicate that the vinyl groups may be neces- 
sary for linking the prosthetic group (iron porphyrin) to the protein 



RESPIRATORY METABOLISM 383 

portion of the cytochrome molecule through a pair of sulphur atoma. 
Lwoff calculated that each flagellate required 520,000 molecules of pro- 
toporhyrin in order to bring the Qoo to 55, and that each organism must 
contain about 700,000 molecules of protoporphyrin before division 
would take place. 

Although cytochrome C was inefi^ective alone, the action of proto- 
porphyrin was increased when cytochrome was present (A. Lwoff, 1936) . 
A. Lwoff (1933) found that the organisms had absorption bands at 
555 and 530 mp, and this indicated the presence of cytochrome. There- 
fore we may conclude that protoporphyrin is necessary for the building 
of ( 1 ) cytochrome or ( 2 ) cytochrome oxidase. Since cytochrome C can- 
not be substituted for protoporphyrin, it seems as if the reaction proto- 
porphyrin -^ porphyrin C is irreversible and that protoporphyrin is neces- 
sary for the synthesis of somethmg other than cytochrome — probably 
cytochrome oxidase. On the assumption that all of the iron is used to 
build the respiratory enzyme, we may calculate the rate of catalysis: one 
gramatom of iron at 28° carries 4.83 grammolecules of O. per second. 
For yeast, Warburg obtained a value of 100. Therefore, on the basis of 
these assumptions, it seems as if the respiratory enzyme of yeast is 20.8 
times as active as that of Sff/gon/onas. It has been demonstrated by M. 
Lwoff (review, A. Lwoff, 1938) that hematin is necessary for the 
growth of 5. 7nuscidaYum, S. cuUcidaruni var. anophelis, L. tropica, L. 
donovani, L. agamae, L. ceramodactyli, and Schizotrypanum cniz'i, as well 
as for the organisms discussed above. The mechanism of its action has 
not been intensively studied, but presumably it may serve a purpose 
similar to that it serves in 5. jasckulata. 

We may conclude from the above experiments that protoporphyrin is 
necessary for the normal metabolism and growth of Strigomonas jascicu- 
lata and that the organism is not capable of its synthesis. Therefore proto- 
porphyrin may be considered a vitamin. Comparable examples are known 
for other organisms: e.g., cholesterol for Trichomonas columhae, T. 
foetus, and Eutrichomastix coluborum; aneurine (vitamin Bj) for 
Glaucoma piriformis, S. oncopelti, S. fasciculata, S. culicidariim, cer- 
tain bacteria, and fungi; pyrimidine and thiazol (parts of the aneurine 
molecule) for Polytomella caeca and Chilomonas Paramecium; ascorbic 
acid for Schizotrypanum cruzi; and lactoflavine, nicotinic acid, and 
phospho-pyridine-nucleotides for various bacteria. In all cases where the 



384 RESPIRATORY METABOLISM 

function of these essential compounds is known or indicated, they seem 
to be necessary for the formation or at least for the normal functioning 
of the respiratory enzymes (review, A. Lwoff, 1938). Recent work on 
the supposed function of most of these substances has been reviewed by 
Burk (1937) and Stern (1938). 

6. THE DETECTION OF OXIDASE, PEROXIDASE, AND CATALASE 

No extensive investigation of isolated enzyme systems of the Protozoa 
has been made. Certain enzymes, especially oxidase and peroxidase, are 
supposed to react with certain stains so that the position of the enzyme 
may be located in the cell. The methods used involve the Nadi, 
Dopa, benzidine-HoOo, and pyronine-anapthol-HoO, reactions (methods 
given by Roskin and Levinsohn, 1926; Guyer, 1936; McClung, 1937). 
These reactions are important in studying vertebrate blood and nerve 
cells, but apparently no correlation has been made of the presence of 
"oxidase" or "peroxidase" granules, detectable by these methods, and 
the respiratory mechanisms discussed above, and it has not been demon- 
strated that these reactions are specific for oxidase or peroxidase. Several 
observations have been made on the Protozoa (Roskin and Levinsohn, 
1926; Bles, 1929), and when more is known about the respiratory 
mechanisms of the Protozoa it might be possible to correlate the results 
of staining and of manometric methods. However, such attempts are 
omitted in the present discussion. Perhaps a certain degree of localization 
of the enzymes could be obtained by centrif uging the organisms and mak- 
ing activity tests on cell fragments, as has been done for peptidase in 
marine ova (Holter, 1936). 

The presence of catalase can be detected by adding hydrogen peroxide 
to a cell suspension and measuring (chemically or manometrically) the 
oxygen evolved. Burge (1924) studied the catalase action of 'Paramecium 
and Colpoda and found that it was decreased by ether and chloroform, 
but not by ethylene or nitrous oxide. A much more accurate method was 
used by Holter and Doyle (1938), who found that the average catalase 
activity per individual of Frontonia, Varamec'mm, and Amoeba was in 
the ratio 190:30:5. Considerable variation was found between different 
cultures and even between different individuals from the same culture. 
Reidmuller (1936) reported only a trace of catalase, no peroxidase, and 
no indophenol oxidase for Trichomonas foetus. 



RESPIRATORY METABOLISM 385 

The Measurement of Anaerobic Metabolism and Glycolysis 

The measurement of anaerobic metabolism is somewhat more complex 
than the measurement of aerobic. The standard criterion of O2 con- 
sumption does not exist, and the auxiliary criterion of CO, production 
indicates only the carbon which is completely oxidized. Sometimes this 
may comprise only a small percentage of the total metabolic changes; 
and in some cases measurements may be complicated by the presence of 
hydrogen, methane, and other gases. Therefore we must usually attempt 
to trace anaerobic metabolism by measuring changes in the concentration 
of several substances in the liquid phase, instead of one or two substances 
in the gaseous phase; and this is more difficult. In some cases it is possible 
to give an organism a known substrate, for example, carbohydrate, and 
to measure the decrease in the quantity of the substrate and the increase 
in the amount of decomposition products at various intervals. From 
carbohydrate decomposition these may be alcohols, aldehydes, and or- 
ganic acids. From protein decomposition we might expect a wide variety 
of nitrogen-containing amino-acid fragments, and by deaminization of 
amino acids a wide variety of organic acids may be produced. From de- 
composition of lipoids we may expect products somewhat similar to 
those from carbohydrates. Methods for the final identification of these 
compounds usually take one into the field of microanalytical biochemistry 
(see Peters and van Slyke, 1932; Friedemann, 1938; and publications 
by von Brand, Reiner, and others, cited below). The identification of 
the acid formed is important in any study of the energetics of anaerobic 
carbohydrate metabolism, because the processes which yield the various 
acids release quite different amounts of energy. For many purposes, 
however, it is customary, if not adequate, to measure acid production by 
manometric measurement of the amount of COo which is released from a 
bicarbonate buffering system, as CO2 or stronger acids are produced by 
the organism. This gives an index of acid production, but leaves us 
ignorant of the nature of the acid. Changes in total titratable acidity or 
alkalinity are also used, and it seems as if, under certain conditions, 
accurate titration curves might be obtained which would give a fair index 
of the kind and amount of acids present. 

In metazoan metabolism it is usually assumed that oxidation of glu- 
cose is preceded by a molecular rearrangement which results in the 
formation of lactic acid. 



386 RESPIRATORY METABOLISM 

Glucose ^ 2 lactic acid -f 43,000 cal. (AH) 
2 lactic acid -^ 60^ -^ 6CO,, -[- 6H2O -\- 634,000 cal. (AH) 

The first reaction is referred to as glycolysis, or cleavage. Glycolysis is 
reversible, and it occurs under both aerobic and anaerobic conditions, but 
the rate of the reverse reaction (lactic acid -^ glucose) is very much less 
under anaerobic than under aerobic conditions. Consequently, in some 
tissues (or in the tissue medium) lactic acid may accumulate aerobically, 
but usually it accumulates only during anaerobiosis. If lactic acid does 
tend to accumulate, it can be measured by allovv'ing it to displace CO2 
from a bicarbonate immersion medium (usually glucose-bicarbonate- 
Ringer). If it is assumed that the CO2 given oi? by oxidation is equal 
to the O, consumed, then the amount of lactic acid can be calculated as 
the "excess CO2," i.e., the COo evolved in addition to that released by 
oxidation. This is expressed as Qo^ for the unit one cmm. of CO, per 
mg. dry weight of tissue per hour. (Older authors use Q%, which is 
easily confused with Qco,, the respiratory COo, and recent German au- 
thors use Qo^ for the same quantity. ) If O2 is replaced by N2, all of the 
COo evolved must come from glycolysis, and the unit is expressed as 
Q^= (or Q^^ or Q^^ ). For comparative studies on various organisms, 
it has been found to be useful to calculate the Meyerhof quotient (M.Q. ) , 
which is defined as 

Qo. 

This is an index of the amount of lactic acid reconverted to glucose per 
unit of oxygen consumption, i.e., a measure of the resynthesis of glucose. 
Recent work on the interpretation of Meyerhof quotients is reviewed by 
Burk (1937). Recent investigations of glycolysis in vertebrate tissue 
indicate that glucose is converted into pyruvic acid, part of which is 
oxidized and part of which is resynthesized into glucose, and that lactic 
acid is a step in the resynthesis, rather than the end product of glycolysis. 
It seems as if this may also be true for Protozoa. 

Occurrence of Anaerobiosis and Glycolysis 

Many Protozoa live in media which are almost if not entirely devoid of 
oxygen. Examples are those which inhabit the bottom of stagnant ponds 



RESPIRATORY METABOLISM 387 

(especially if a considerable amount of decaying organic matter, and 
consequently hydrogen sulphide, is present), those which are found in 
sewage-disposal plants, those which appear near the bottom of putrid 
laboratory cultures, and those which inhabit the lumen of the lower in- 
testine of Metazoa. These organisms, because of the characteristics of 
their environment, are deprived of one of the chief sources of energy 
available to other animals — the reduction of molecular oxygen, and they 
must be able to obtain energy by other methods, such as molecular re- 
arrangements (e.g., glucose to lactic acid) or oxido-reductions (e.g., 
glucose to CO2 and alcohol). A summary of the early theories of an- 
aerobic fermentations is given by Slater (1928), and a review of the 
data pertaining to anaerobic life of Protozoa and other invertebrates is 
given by von Brand ( 1934) . Some of the anaerobic Protozoa seem to be 
obligatory anaerobes and are quickly killed by aeration (e.g., Trepomonas 
agilis, Lackey, 1932). Therefore one might expect them to have a type 
of metabolism comparable to those of the anaerobic bacteria. 

Other organisms, such as certain intestinal forms, are certainly not 
strict anaerobes, but are facultative, or amphibiotic. Measurements of the 
intestinal gases (reviewed by von Brand and Jahn, 1940) and of the oxi- 
dation-reduction potential of the digestive tract (Jahn, 1933a) indicate 
that the lumen of the intestinal tract is largely devoid of oxygen. How- 
ever, organisms which live at the surface of the epithelium (e.g., Giar- 
dia) and within the villi, and especially those such as Endamoeha histo- 
lytica and Balantidium coli which invade the tissue, do have access to 
molecular oxygen. The O. tension of the environment of the rumen in- 
fusoria of ruminants must be extremely variable, but, for considerable pe- 
riods of time, almost devoid of oxygen. The question then arises as to 
what kind or kinds of respiratory mechanisms are present in these faculta- 
tive organisms. The same question arises with such organisms as Para- 
meciuni, which are normally aerobic but can withstand lack of oxygen 
for a relatively long period of time. Do the facultative anaerobes of the 
phylum Protozoa have respiratory mechanisms comparable to those of 
bacterial facultative anaerobes? This question, although interesting and 
suggestive, is unanswerable at present because we know nothing about 
the respiratory mechanisms of anaerobic Protozoa, and not very much 
about those of bacteria. However, recent investigations indicate that 
among bacteria the respiratory mechanism of the strict anaerobes is 



388 RESPIRATORY METABOLISM 

probably different from the anaerobic mechanism of the facultative an- 
aerobes (Broh-Kahn and Mirsky, 1938). 

There is considerable evidence that carbohydrate decomposition takes 
place in Protozoa under anaerobic conditions. It was found by Putter 
(1905) that the glycogen content of Paramecium decreased under an- 
aerobic conditions. He also found that Paramecium poor in glycogen 
could live anaerobically for a considerable length of time, probably at 
the expense of albumen. A. Lwoff (1932) found that Glaucoma piri- 
formis could live three days without oxygen only if sugar were present. 
M. Lwoff (1934) obtained a value of 10 for the Q^^ of G. piriformis in 
peptone broth (Qoo = 35). Emerson (1929) found that under an- 
aerobic conditions 80 mm^ of Blepharistna released 12.5 mm.^ CO2 per 
hour from a bicarbonate buffer mixture; negative results were obtained 
with Amoeba proteus. Zhinkin (1930) demonstrated that the glycogen 
content of Stentor decreased under anaerobic conditions and that visible 
fat increased. Upon exposure to Oo that fat disappeared. This apparent 
conversion of glycogen to fat was observed in experimental cultures and 
under natural conditions in winter when the O, content of ponds was 
negligible, but it did not occur in experimental cultures in the presence 
of light because of the photosynthetic action of zoochlorellae. Some data 
of this type was also obtained for Prorodon teres and Loxodes (Zhinkin, 
1930) and for Paramecium (Pacinotti, 1914). The possible changes 
which take place in the glycogen content of intestinal amoebae and 
ciliates should also be investigated in this connection (see discussion by 
von Brand, 1934). 

The trypanosomes are a group of organisms which live under con- 
ditions of high Oo tension, but they apparently have a high degree of 
anaerobic metabolism (glycolysis). At least, they use much more sugar 
than they could possibly oxidize with the Oo which they consume, and 
apparently the amount of acid produced by glucose destruction does not 
differ much under aerobic or anaerobic conditions. According to the data 
of von Fenyvessy and Reiner (1924), the O2 consumption for a billion 
trypanosomes (T. equiperdum) suspended in diluted blood was about 
0.07 mg. per hour. The sugar consumed under similar conditions (Yorke, 
Adams, and Murgatroyd, 1929) was about 5 mg. Since complete oxida- 
tion of 5 mg. of sugar requires about 5 mg. of Oo, it appears as if the 
major portion of the sugar destruction was anaerobic. This is discussed 



RESPIRATORY METABOLISM 389 

by von Brand (1934, 1935). On the basis of an assumed R.Q. of one, 
von Fenyvessy and Reiner (1928) subtracted the Qo, from the rate of 
CO2 evolution from a bicarbonate solution and found that the resulting 
value was equal to the Qn» . Therefore the rate of glycolysis (acid pro- 
duction) is the same in O. or N. and is independent of O, consumption. 
The data of von Fenyvessy and Reiner showed that the amount of CO2 
evolved when the organisms were in bicarbonate-glucose-Ringer was so 
high under aerobic conditions that apparent R.Q. values of 1.7 to 3.6 
were obtained. Therefore we must conclude that glucose -^ acid conver- 
sion is very high in T. equiperdum. According to the experiments of von 
Brand, Regendanz, and Weise (1932), this acid production is appar- 
ently not a true glycolysis, because lactic acid could not be detected in the 
medium by chemical methods. However, if we consider lactic acid to be 
a step in the resyn thesis of pyruvic acid to glucose (see above), the ab- 
sence of lactic acid may merely mean that resynthesis does not occur. In 
this case the Meyerhof quotient should be zero. It was shown by Reiner 
and Smythe (1934) and Reiner, Smythe, and Pedlow (1936) that 
aerobic sugar destruction by T. equiperdum was as follows: 

1 glucose -> 1 glycerol -[- 1 pyruvic acid 
1 glycerol _|- O, -^ 1 pyruvic acid -]- 2HoO 

Apparently lactic acid and CO^ were not produced. For T. letvisi, under 
aerobic conditions, the end products were identified as succinic, acetic, 
and formic acids, ethyl alcohol, and carbon dioxide. 

It has been suggested that the large amount of acid produced by 
trypanosomes might be the mechanism by which toxic effects are pro- 
duced. This possibility was investigated for T. evansi by Kligler, Geiger, 
and Comaroff (1929), who analyzed the blood of infected rats and 
concluded that death was caused by lactic acid acidosis. In subsequent 
publications (Kligler, Geiger, and Comaroff, 1930; Geiger, Kligler, and 
Comaroff, 1930) they reported the measurement of glycolysis of T. 
evansi, having obtained even higher values (9.2 mgm. /billion/hour) 
than Yorke, Adams, and Murgatroyd had obtained for T. equiperdum. 
Von Brand, Regendanz, and Weise (1932) measured the glucose, lactic 
acid, and alkali reserve of animals infected with T. gambiense, T. brucei, 
and T. equiperdum, and found no evidence of low glucose, high lactic 
acid, or low alkali reserves, and therefore no support for the acidosis 



390 RESPIRATORY METABOLISM 

theory of death. Von Brand (1933) measured the rate of sugar destruc- 
tion for various trypanosomes, and obtained high values (8.0 mgm. /bil- 
lion/hour) for the pathogenic trypanosomes T. hrucei, T. gmnbiense, 
T. rhodesiense, and T. congolense, and very low values for the nonpatho- 
genic T. letvisi (about 1.4), and still lower values for the pathogenic 
SchJzotrypanuni cruzi. The results of these investigators indicate that 
although sugar destruction and formation of acid may be a contributing 
factor, it will not explain all of the pathological effects of the trypano- 
somes. This is reviewed by von Brand (1938). 

It is interesting to compare the high aerobic glycolysis rate of trypano- 
somes with that of malignant tumors. The Warburg quotient (aerobic 
glycolysis/Qoo ) for normal tissues is usually less than 0.3 (except retina 
and placenta), while that for benign tumors is about one, and that for 
malignant tumors is 3.1-3.9 (review, Needham, 1931). The Warburg 
quotients calculated from the data of von Fenyvessy and Reiner (1928) 
for T. equiperdum are 0.78 to 2.67. It has been found that KCN may 
change the Warburg quotient of chick embryos from 0.1 to 3.4, but 
the quotient for T. equiperdum, in the presence of KCN, showed no 
significant change (0.78 and 0.80 in two experiments). These compari- 
sons may be taken to indicate that the relative glycolytic rate of the 
trypanosomes is different from that of normal tissues and resembles in 
certain respects that of benign or malignant tumors, but the fact that 
pyruvic and other acids are formed by trypanosomes instead of lactic 
acid invalidates this comparison. 

Meyerhof quotients were calculated by A. Lwoff (1933) for Stri- 
gomonas jasckulatn, S. oncopelti, and heptonionas ctenocephali and 
were found to be 1.20, 1.38, and 0.125 respectively. The first two are 
within the normal range of metazoan tissues (Needham, 1931), but 
that of Leptowonas is very low. Values of the M.Q. calculated from the 
data of von Fenyvessy and Reiner (1928) on trypanosomes are approxi- 
mately zero, indicating no reversal of glycolysis, and this conclusion 
agrees with the chemical equations given above. 

Why Are Anaerobes Anaerobes, and Aerobes Aerobes.^ 

One question which arises in any treatment of anaerobiosis is, "Why 
does oxygen prevent growth of obligatory anaerobes?" There are several 
explanatory theories: 



RESPIRATORY METABOLISM 391 

1. Oxygen is directly lethal to the cell. 

2. Anaerobes do not contain catalase and therefore are incapable of 
destroying the toxic HoO^ which is formed by reduction of oxygen (see 
equations given above). 

3. Growth of anaerobes is dependent upon the presence of a low 
oxidation-reduction potential in the medium, the attainment of which 
is prevented by oxygen. 

4. Oo forms a loose chemical complex with the respiratory system of 
obligatory anaerobes, and thereby inhibits its activity. 

The relative merits of these theories, as applied to bacteria, are dis- 
cussed by Hewitt (1936) and Broh-Kahn and Mirsky (1938). The first 
theory is certainly not true for those anaerobic organisms which will 
grow under anaerobic conditions after exposure to oxygen. The second 
theory is supported by considerable evidence, in that most anaerobes do 
not contain catalase, and in that some bacteria (e.g., pneumococci) will 
grow aerobically until they are killed by the accumulation of HgOo re- 
sulting from their metabolism (so-called "suicide" of cultures). How- 
ever, some anaerobes do contain catalase, and apparently it has not been 
definitely demonstrated that strict anaerobes consume O, in order to 
produce HgO,, or even that obligatory anaerobes do produce H.Og (Broh- 
Kahn and Mirsky). The theory, however, might still be applicable to 
organisms such as penumococci and hemolytic streptococci, and to 
Escherichia coli in the presence of HCN and methylene blue. In these 
cases appreciable amounts of HgO, can be detected. 

Among the Protozoa we have very little evidence of the relative merits 
of the first two of these theories. It is shown by the work of Cleveland 
on termites and on xylophagous cockroaches (Cleveland, Hall, Sanders, 
and Collier, 1934, include citations of earlier papers) that the symbiotic 
Protozoa which inhabit the digestive tracts of these organisms are prob- 
ably strict anaerobes. At least the O2 tension of their normal environment 
is extremely low, and they are rapidly killed by appreciable quantities 
of molecular O.,. Cleveland found that at 23° C. the time necessary for 
death of the symbionts of termites was an inverse function of oxygen 
tension (e.g., in Termopsis, all Protozoa were dead in 72 hours at one 
atmosphere, in 30 minutes at 3.5 atmospheres). This is apparently due 
to an increase in Oo concentration in the digestive tract, with increased 
O2 pressure in the atmosphere, and this could easily be explained on the 



392 RESPIRATORY METABOLISM 

basis of either of the above two theories. Additional evidence, however, 
might be gained for the second theory from the fact that one atmosphere 
of O2 is more toxic at low temperatures (4-5° C.) than at high (23- 
25° C.) , and that four atmospheres of Oo are more toxic at high tempera- 
tures than at low. The greater solubility of O2 at 4-5° C. can account for 
the greater toxic effect with one atmosphere pressure. However, the re- 
verse effect at four atmospheres O2 pressure must, as suggested by Cleve- 
land, be connected in some manner with metabolic processes. Super- 
ficially, at least, these results seem to be explicable on the basis of the 
second of the above theories, i.e., the organisms grew more rapidly and 
produced more H0O2 at the higher temperatures. An examination of the 
protozoa for catalase, or of the digestive contents of oxygenated insects 
for H2O2, might yield pertinent information. 

The data of Cleveland (1925) on the toxicity of oxygen for the in- 
testinal Protozoa of earthworms, salamanders, frogs, and goldfish, are 
possibly open to this explanation, but here also we lack experimental 
evidence. Where such defaunation procedures failed, as in the rat, we 
might assume that the O2 tension of the digestive tract was not raised 
in a manner comparable with that which occurred in the smaller or- 
ganisms, or that the Protozoa present were more resistant. The former 
theory seems much more probable. (For review of the chemistry of the 
intestinal contents, see von Brand and Jahn, 1940.) 

The theory that the growth of anaerobes is dependent upon a low 
oxidation-reduction potential in the medium was proposed by Quastel 
and Stephenson (1926), and has gained considerable support among 
bacteriologists (review, Hewitt, 1936) and some dissent (literature 
cited by Broh-Kahn and Mirsky, 1938) . Positive evidence consists mainly 
of the facts that (1) during the growth phases of anaerobic cultures, 
especially the sporulating anaerobes, much lower oxidation-reduction 
potentials are produced than during the growth phases of cultures of 
aerobes; and (2) anaerobic forms do not start growing until the 
potential is quite low (Eh < -f- 100 mv.). The first type of evidence 
does not help to distinguish between cause and effect, and there are 
some exceptions to the general trend. Most of these, however, are due 
to the fact that the organism is only one factor which tends to determine 
the Eh of the medium; the chemical composition of the medium certainly 
determines, to a great extent, what potentials may be attained. The second 



RESPIRATORY METABOLISM 393 

type of evidence is well founded in fact — anaerobes do not grow in media 
of high Eh value. However, if the Ei, value of a suitable medium is low- 
ered through displacement of air with Ho or N,, or by various chemical 
reagents, or by the growth of an aerobic organism, then the anaerobic 
forms are capable of growth. According to this theory, anaerobes and 
aerobes differ in their ability to grow at various points along the Eh scale, 
in a manner comparable to that which is exhibited by various acido- 
philic and basiphilic forms in growing at various points along the pH 
scale. Of course, there are intermediate-range and wide-range forms in 
respect to both pH and Eh. The fact that the toxic effects of lack of O2, 
or of supernormal Oo tensions, are not equal in all species supports this 
idea, but these data, of course, are subject to other interpretations. 

Investigations of the role of oxidation-reduction potentials among the 
Protozoa have never passed the preliminary stages. The possible im- 
portance of such a study was pointed out by Jahn (1933b, 1934), in 
connection with experiments on -SH compounds, on the toxic action of 
methylene blue and on possible relationships with auto- and allelocataly- 
sis. Measurements of the Eh of Chilomonas cultures (Jahn, 1935b), of 
hay infusions (Efimoff, Nekrassow, and Efimoff, 1928), and of digestive 
contents (Jahn, 1933a) have been made. However, until more data be- 
come available, most of these results are difficult to interpret. It was de- 
termined that Chilomonas would grow in mixtures of NaSH and HjOo 
only if the concentrations of these were balanced so that the medium 
just failed to reduce methylene blue. This might indicate a microaero- 
philic tendency for Chilomonas. Other experiments with Chilomonas 
indicated that it could live, but could grow only slowly, however, in 
media in which methylene blue was reduced. Neither of these ideas is 
contradictory to its known habits in laboratory cultures. It was also 
demonstrated (Jahn, 1935b) that casein-acetate broth cultures of Chi- 
lomonas, when exposed to the air, developed potentials of -20 mv. at 
pH 7.55, a point at which methylene blue is about half reduced. The 
chief difficulty in interpreting experiments pertaining to the effect of Eh 
on growth is that it is necessary to change Oo tension in order to change 
Eh. This makes an experiment containing only one variable seemingly im- 
possible to execute, and the theory, therefore, has not been amenable 
to experimental approach. 

The fourth theory of the effect of O. on anaerobes — that of an inac- 



394 RESPIRATORY METABOLISM 

tivation of the respiratory mechanism of obligatory anaerobes by O^, — 
is mentioned by Broh-Kahn and Mirsky (1938), but at present is 
unsupported by experimental evidence. The bacteriostatic effect of dyes 
might also be interpreted to mean that these have an inactivating effect 
on the respiratory mechanism. 

In connection with the effect of oxygen on anaerobes, it should be 
mentioned that the converse problem also exists. Why do aerobes die in 
the absence of oxygen.'' On this question there has been considerably 
less discussion in the literature than on the former. It can be seen from 
the respiratory equations given above that activation of the substrate 
and partial oxidation can proceed without Oo. Even in the presence of 
O, a considerable destruction of substrate, in some forms (e.g., trypano- 
somes, cited above), seems to be incomplete. Poisoning of the aerobic 
cell under anaerobic conditions is supposedly caused by the accumulation 
of toxic products of carbohydrate cleavage or of incomplete oxidation, 
or by the conversion of all of the respiratory pigment to the reduced 
state. In facultative anaerobes the former, and in obligatory aerobes the 
latter theory seems more probable. The observation of Faure-Fremiet, 
Leon, Mayer, and Plantefol (1929) that Paranieciuni withstands lack of 
O2 longer at 4° C. than at higher temperatures, is open to either inter- 
pretation. The data of Piitter (1905), which indicate that 'Paramecium 
can live longer under anaerobic conditions when the ratio of volume of 
medium to cells is higher, can be explained on the basis of accumulation 
of toxic products. What these toxic products are probably depends to a 
large extent upon the organism, the substrate, and the conditions of the 
experiment, and the most likely possibilities include lactic and lower 
fatty acids. 

Oxidation-Reduction Potential Versus Respiration 
AND Growth 

Another question which arises is why we might or might not expect 
the oxidation-reduction potential of the medium to affect the respiration 
and growth of the organism. It is obvious from the outline of the respir- 
atory processes given above, and from many other types of data, that 
oxidation-reduction phenomena are involved in respiration. The respira- 
tory pigments and perhaps also the respiratory enzymes are reversible 
oxidation-reduction systems. Therefore their action should be affected 



RESPIRATORY METABOLISM 395 

by the potential of the medium in which they are found, that is, by the 
oxidation-reduction potential of protoplasm, and they, in turn, must 
to a large extent determine this potential. If we were to mix numerous 
half-reduced, completely reversible oxidation-reduction indicators in a 
homogeneous solution, an equilibrium would be reached, and the po- 
tential attained would depend upon the £„ values of the substances and 
upon their relative amounts. Substances with Eo values far from the result- 
ing Eh value of the solution would be either completely reduced or com- 
pletely oxidized, and would be unable to contribute much toward oxidizing 
or reducing small amounts of added materials unless the Eu of the mixture 
were appreciably changed. 

Obviously such a simple system is not present in protoplasm. Because 
of the presence of irreversible systems and of the continual introduction 
of new substrate and the removal of certain end products, a true equi- 
librium is never attained. Also, the colloidal nature of protoplasm makes 
possible the existence of different Ei, values in different phases of the 
substance, and the differential adsorption of oxidized and reduced ma- 
terial at interfaces may produce a potential different from that in any of 
the phases. Therefore the term "oxidation-reduction potential of proto- 
plasm" may be without any interpretable significance (for discussion, see 
Jahn, 1934; Korr, 1938). But there must certainly be a significance to 
the Eo values of the respiratory pigments, and the possibility of an indi- 
vidual expression of these values may be maintained by the polyphasic 
nature of protoplasm. The oxidation-reduction potential, which can be 
measured with indicators, is probably an index of the potential developed 
by one or more of these pigments (for summary of such measurements, 
see Chambers, 1933; Cohen, 1933). It is known that the apparent Ei> 
value of protoplasm, as measured by indicators, varies with the Eh of the 
external medium when the external O., tension is changed. Therefore, 
why cannot the Ei, of the external medium determine the degree of re- 
duction of the respiratory pigments and therefore the rate of respiration.'' 
This mechanism might be used to explain the inhibition which is pro- 
duced by oxidation-reduction indicators in cultures of bacteria (Dubos, 
1929) and in cultures of Chilomonas (Jahn, 1933b). One difficulty in 
predicting what reactions would occur in protoplasm, even if we had a 
thorough knowledge of the oxidation-reduction systems involved, is the 
fact that such knowledge can tell us only what reactions might or might 



396 RESPIRATORY METABOLISM 

not occur if all of the reactions were reversible. Since many of the re- 
acting substances are changed irreversibly and since the rates of reactions 
are dependent not only upon Eo values but upon enzymes, knowledge of 
Eo and Eh values cannot indicate what reactions will occur. 

Much of this discussion of oxidation-reduction potentials is pure 
speculation, but it is the type of speculation (often unexpressed) which 
has spurred investigators to a study of the naturally occurring oxidation- 
reduction systems, of the apparent oxidation-reduction potential of proto- 
plasm, and of the Eh values developed in bacterial and protozoan cultures 
(reviews, Needham and Needham, 1927; Wurmser, 1932; Chambers, 
1933; Clark, 1934; Hewitt, 1936). Interpretations of the data have not 
always been as fruitful as one might expect, and one is led at times to 
suspect that the modes, if not the points of attack on the problem, are in 
need of revision. However, since the necessity of some such relationship 
as outlined above seems sound, it is more probable that merely the time 
for the harvest has not yet arrived. Clark (1934) estimated that another 
half century will be necessary for the solution of these problems. 

Another means by which the oxidation-reduction potential of the 
medium is supposed to affect metabolism is described by the surface catal- 
ysis theory suggested by Quastel (1930), Kluyver (1931), and others 
(discussed by Hewitt, 1936). It is suggested that many oxidative pro- 
cesses of bacteria take place at the surface of the cell (Quastel, 1930) , and 
it seems as if for these reactions the Eh of the medium would be more im- 
portant than that of the protoplasm. It is also very probable that oxida- 
tion-reduction enzymes are merely surface catalysts, which produce their 
effect by nature of intense interfacial electrical fields (Kluyver, 1931). 
These fields might be affected by the potential of the medium, whether 
they occur at the cell surface or within the protoplasm. An intriguing 
speculation would be that the respiratory pigments are distributed among 
the various phases of protoplasm, and that the enzymes are actually the 
interfaces of the emulsion. Unfortunately, such ideas are difficult to 
check experimentally. However, since oxidation-reduction enzymes are 
proteins which in all probability exert their catalytic properties through 
surface action, it is possible that the catalytic interfaces of the cells, dis- 
cussed by Kluyver (1931), are merely the surfaces of the protein mole- 
cules. 



RESPIRATORY METABOLISM 397 

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Zhinkin, L. M. 1930. Zur Frage der Reservestoflfe bei Infusorien. Z. Morph. 
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CHAPTER VII 

THE CONTRACTILE VACUOLE 
J. H. Weatherby 

Introduction 

Since the first description of the protozoan contractile vacuole, prob- 
ably made by Spallanzani in 1776, few structures in these organisms have 
received such intensive investigation. Unfortunately, solutions of many 
of the perplexing questions which have arisen as a result of these studies 
are not yet at hand. Indeed, much of the more recent work has given rise 
to entirely new questions which are no less insistent in their demands 
for answers than were the earlier ones. In the literature claims and 
counterclaims are abundant; important discoveries have been made only 
to be discarded because of lack of confirmation, or, in some instances, 
because of direct contradiction. In view of the somewhat confused state 
of the evidence concerning contractile vacuoles a re-survey of some of 
the more important questions seems to be in order. 

Probably the first question asked by the first investigator to see a con- 
tractile vacuole was "What is its function?" Needless to say, this first 
investigator did not learn the answer, and, in the opinions of many, the 
most recent investigation probably does not supply the complete answer. 
Following this question there have been others hardly less interesting. 
Is it essential to life? Is it a permanent structure, or does it arise anew 
at the beginning of each new cycle? Does it always occupy the same 
position in the organism with respect to other structures? Is the vacuole 
surrounded by a permanent membrane? Is its discharge to the exterior 
through a permanent excretory pore? If there is no pre-formed excretory 
pore, how may one explain the formation even of a temporary pore, and 
once formed how is it closed again? What natural forces operate to expel 
the contents of the vacuole? There are many other equally interesting 
questions, but only a few can be considered at this time. 

The most promising order for discussing these problems seems to be 



THE CONTRACTILE VACUOLE 405 

to deal first with those pertaining to origin of the vacuole — to see, if 
possible, just where this organelle comes from, if it does not exist in 
the cell as a permanent structure. Then, having traced its origin, questions 
dealing with structure and function will follow in more logical order. 
An attempt will be made to follow this general plan, but the very nature 
of the subject will necessitate digressions from time to time. 

While a conscientious review of the literature has been attempted, it 
is quite possible that important publications have been overlooked. It is 
hoped that this will prove not to be true, not only for the sake of com- 
pleteness, but also for the sake of giving credit where credit is due. The 
author herewith offers his apologies to anyone whose labors have not 
been acknowledged. 

The Origin of Contractile Vacuoles 

Metcalf (1910) noticed in amoeba of the proteus type that the vacu- 
ole is surrounded by a layer of granules of the same approximate size 
and appearance as the "microsomes" of the general cytoplasm. When 
the vacuole is of moderate size, these granules form a layer on its surface 
one granule thick; when the vacuole is fully distended, as just before 
systole, there are spaces between the granules; but when the vacuole is 
small the layer may be several granules thick. At systole the vacuole 
usually collapses completely, and the granules may be seen clumped to- 
gether in the region of the cytoplasm previously occupied by the vacuole. 
The new vacuole arises in the midst of these granules, and is formed by 
the fusion of several small vacuoles. According to Metcalf, who reported 
observations which sometimes lasted for as long as several hours on a 
single organism, the vacuole never arises in any other part of the body 
under normal conditions, except among the granules which surrounded 
it before its last contraction. From these observations he concludes that 
the granules are associated in some way with the origin and the function 
of the vacuole, and for this reason calls them "excretory granules." How- 
ever, he decides that the granules are not essential for life, since most 
of them, together with the vacuole, may be removed from an Amoeba 
by operation without a fatal result. Under these conditions a new vacuole 
develops, although there are few if any granules to be seen surrounding 
it when it first appears. Metcalf reaffirmed his statement concerning these 
observations in 1926. 



406 THE CONTRACTILE VACUOLE 

Mast (1926) agrees with Metcalf concerning the frequent presence 
of granules around the vacuole, but does not interpret this as indicating 
a physiological association between them. This opinion is based on his 
having observed vacuoles functioning perfectly normally without the 
presence of a single granule in the immediate vicinity of the vacuole. 
To these granules Mast applies the name "beta granules," to distinguish 
them from others of a different nature which are also present in the 
cytoplasm. Mast and Doyle (1935) reinvestigated the relationship be- 
tween granules and vacuole. By centrifuging amoebae it is possible to 
cause stratification of various cytoplasmic constituents. Organisms treated 
in such a manner can be operated on so as to remove all or any desired 
portion of almost any one of the constituents, including these granules. 
It was found by Mast and Doyle that removal of all or most of the gran- 
ules resulted in the death of the organism. Removal of fewer granules 
caused a decrease in pulsation frequency of the vacuole, which was di- 
rectly proportional to the relative number of granules removed; that is, 
pulsation frequency was found to be directly proportional to the number 
of granules remaining. Removal of the contractile vacuole alone resulted 
in the prompt formation of another. Concerning this same question, 
Mast (1938, p. 312) more recently states : 

The beta granules around the contractile vacuole vary greatly in number 
and the layer of substance in which they are embedded varies greatly in 
thickness, without any apparent variation in the function of the vacuole. 
These facts indicate that neither the granules nor the layer of substance is 
involved in the function of the contractile vacuole, at least not directly. 

Howland (1924a) found that there is no concentration of granules 
on the surface of the vacuole in Amoeba verrucosa, but she considers 
it likely that the vacuole arises from the coalescing of small hyaline 
globules, which in turn are derived from the dissolving of granules. 
In any case, Howland traces the ultimate origin of the vacuole back to 
granules in somewhat the same manner that Metcalf does, although in 
A. verrucosa these granules are probably dispersed throughout the cyto- 
plasm. On the other hand, Haye (1930) found in fixed and stained 
preparations of A. vers pert iUo essentially the same relationship between 
granules and vacuole as described by Metcalf and later by Mast; that is, 
the filled vacuole is more or less covered by granules, and after systole 
the new vacuole arises in the midst of these granules. 



THE CONTRACTILE VACUOLE 407 

Hall (1930a) studied the cytoplasmic inclusions in Trichamoeba after 
osmic and silver impregnation. In a few instances he observed the adher- 
ence of blackened globules to the outer surfaces of vacuoles. At first 
glance these appeared to be vacuoles with heavily impregnated walls, 
but close observation revealed the granular or globular nature of the 
blackened material. 

It must be remembered that these granules are not confined to the im- 
mediate vicinity of the contractile vacuole, but usually are scattered 
throughout the entire cytoplasm as well. If the origin of the vacuole is 
associated with and dependent on the presence of these granules, then one 
would expect other parts of the organism to be at least potentially capable 
of giving rise to vacuoles, since some granules are present in other 
parts. That such a phenomenon actually occurs in Amoeba has been ob- 
served by various authors, among whom are Day (1927), as well as 
Howland and Mast and Doyle. In this connection it is interesting to note 
that Dimitrowa (1928) was able to induce formation of extra vacuoles 
in Paramecium caudatum by interfering mechanically with the normal 
function of those already present. These extra vacuoles usually appeared 
to be entirely normal, although in a few instances there were no radial 
canals. The customary number of vacuoles was restored at fission by 
failure of the organism to form new ones if two extra ones had been in- 
duced, or by the formation of one new vacuole if only one had been in- 
duced artificially. In the event that there were three extra vacuoles, one 
daughter cell received three and formed a single new one when it in turn 
divided. 

Haye (1930) investigated eight species from two orders of flagellates. 
In Phacus pleuronectes he found that the walls of contractile vacuoles 
contain lipoid granules which are arranged in a net-like fashion. In 
Euglena piscijormis and Tracbelomonas hispida the surfaces of both the 
reservoir and the vacuoles show a granular structure. In Pevidinium 
ste'mii no granules were observed, nor were accessory vacuoles seen, ex- 
cept in organisms obtained from a laboratory aquarium in which con- 
ditions were thought to have been abnormal. A differentiated plasma 
zone, reminding one of the "excretory plasma" of fresh-water Protozoa, 
was noted around the pusule. In the wall of the pusule of P. divergens 
were observed lipoid granules similar to those in the wall of the contrac- 
tile vacuole of fresh-water Protozoa. Besides the two sac pusules, a col- 



408 THE CONTRACTILE VACUOLE 

lecting pusule with daughter vacuoles and numerous accessory vacuoles 
were observed in Phalacroma sp. In Goniodoma sp. there were, besides 
a sac pusule, a collecting pusule with daughter pusules and an accessory 
vacuole. Only one large pusule was noted in Ceratium hirnndineUa. Ir^ 
both orders of flagellates Haye believes that emptying of accessory vacu- 
oles is accomplished by diffusion through the walls into the contractile 
vacuole, rather than by coalescence with it. 

Hall (1930b) found that in Menoidium, stained according to the Da 

\ Fano silver method, the contractile vacuole is formed by the fusion of 
several smaller vacuoles arising near the gullet. 

The mode of origin of contractile vacuoles has been studied in a 
greater variety of ciliates than in either rhizopods or flagellates, and in- 
formation on this subject is proportionally more abundant. Taylor 
(1923) observed in Euplotes that the vacuole (VJ, in its final form 
immediately before contraction, is the result of the fusion of several 
smaller vacuoles, and that these smaller vacuoles (designated as group 
V2) in turn are formed by the fusion of still smaller vacuoles (group 
V3) . The smallest vacuoles in the series are thought to arise as the result 
of the dissolving of granules, or to arise de novo. Thus Taylor suggests 
granules as a possible source of vacuolar fluid, and he observed formation 
of the vacuole by the fusion of several small accessory vacuoles. King 
( 1933) , who studied Euplotes after impregnation with osmic acid, found 

.that the smallest visible accessory vacuoles (Vg) have their origin at the 
distal ends of a very large number of collecting canals, located just under 
the ectoplasm on the dorsal surface of the ciliate. These canals radiate 
like a sun-burst from the vicinity of the vacuoles, and seem to end blindly 
in the protoplasm of the organism. These canals have a diameter of ap- 
proximately 0.5 micron at their distal ends, and become relatively much 
narrower as they pass away from the region of the vacuoles. The canals 
are not visible in living organisms, but may be clearly demonstrated by 
proper impregnation with osmic acid. On the basis of information now 
available, it is difficult to tell whether the canals described by King and 
the granules mentioned by Taylor represent different interpretations of 
the same structures, observed under different conditions, or whether the 
canals merely provide a means for the transport of fluids which have 
originated in more distant parts of the body as a result of the activity 
of granules. 



THE CONTRACTILE VACUOLE 409 

Of particular interest are the observations of MacLennan (1933) on 
the Ophryoscolecidae, cihates from the stomachs of cattle. The cycle of 
the contractile vacuole was studied in both living and fixed material, in- 
cluding the following genera: Ophryoscolex, Epidinium, Ostracodinuim, 
Poly plastron, Eudiplodinium, and Metadinmm. In all these genera the 
contractile vacuole is formed by the coalescence of small accessory vacu- 
oles, just as in Eu plates, as described by Taylor and also by King, and 
in Amoeba, as described by Metcalf . These accessory vacuoles arise from 
the dissolving of granules which are found in sharply defined regions 
around the contractile vacuole in Eudiplodinium and Metadinium, in a 
narrow dorsal strip of the ectoplasm in Ostracodinium, and in the whole 
ectoplasm in Ophryoscolex and Epidinium. If one may be permitted to 
assume that the canals and granules in Euplotes are identical, then the 
mode of origin of the contractile vacuole in this form is quite similar to 
that described by MacLennan for the Ophryoscolecidae; and in its funda- 
mental features it also resembles that reported for amoebae as well as for 
some of the flagellates. 

In addition to the flagellates previously mentioned, Haye (1930) also 
studied representatives of thirteen genera from four orders of ciliates. 
In Opalina dimidiata, Isotricha prostoma, Spirostomum ambiguum, and 
Nyctotherus cordiformis little was observed which suggests the mode 
of origin of contractile vacuoles. Except for the fact that the walls of 
the canals were found to contain lighter and darker zones — probably 
because of the presence of lipoid granules — little was observed in Para- 
mecium caudatum which may be associated with origin of vacuoles or 
their contents. Rod-shaped entosomes were found closely packed about 
the wall of the vacuole in Lionotus fasciola. In Stentor polymorpha the 
wall of the vacuole is very delicate and shows only here and there a 
granular structure; several secondary vacuoles are usually present. In 
Blepharisma undulans, Balantidium entozoon, Polyplastron multivesi- 
culatum, Ostracodinium gracile, and Ophrydium versatile are to be found 
granules (entosomes) within the wall, or closely associated with the 
wall, of the contractile vacuole. The vacuole in Epistylis plicatilis is 
formed from numerous secondary vacuoles; no granules or entosomes are 
to be observed. 

Von Gelei (1933) states that the vacuole system in Spathidium con- 
sists of a primary vacuole, located usually in the posterior end of the 



410 THE CONTRACTILE VACUOLE 

organism. Around this are one or two rows of smaller secondary vacuoles, 
which fuse and give rise to a new primary vacuole following systole. 
Whether or not these secondary vacuoles originate from granules was 
not ascertained by von Gelei. Essentially the same relationship between 
primary and secondary vacuoles in Blepharisma was described by Moore 
(1934), who made the further statement that excretory granules could 
not be observed. Both Wenrich (1926) and King (1928) found the 
vacuoles of P. trichium to be vesicle-fed, although neither author men- 
tioned the origin of these vesicles. Day (1930) found that the vacuolar 
fluid reaches the elongated canal of Spirostomiim by the fusion of small 
vacuoles with the canal throughout its entire length. A similar source 
of fluid in canals of P. caudatum was also reported, but in neither in- 
stance was the origin of the accessory vacuoles mentioned. 

Faure-Fremiet (1925) observed the filling of contractile vacuoles in 
several species of Vorticella by the discharge of small vesicles into the 
vacuole. These vesicles originate in the wall of the vacuole, and cor- 
respond to the "mural vacuoles" described by Haye (1930) for Cam- 
panella. Chilodon, Dogielella, and some of the Ophryoscolecidae. Nas- 
sonov (1925) also investigated Chilodon and Dogielella, and found 
structure and mechanism of filling to be somewhat difl^erent from that 
described by Haye. According to Nassonov, vacuoles in these forms do 
not appear to have the thick walls described by Haye, nor even to have 
any sort of membrane, but lie directly in the cytoplasm. However, there 
is a strongly osmiophilic structure closely associated with them, which, 
for Chilodon at least, and possibly also for Dogielella, may be mistaken 
for a thick wall or membrane under certain conditions. In both forms the 
osmiophilic structure remains essentially unaltered in appearance after 
collapse of the vacuole. Nassonov observed the origin of accessory 
vacuoles (the mural vacuoles of Haye) in these osmiophilic structures, 
and believes them to contribute to the filling of the contractile vacuole. 

Many authors hold that in certain Protozoa, typified by Paramecium 
caudatum, the question of origin of the contractile vacuole does not arise, 
since in these forms the vacuole system is a permanent structure. This 
view is not universally accepted, as will be pointed out later. But, whether 
permanent or temporary, there still exists a no less fundamental question 
as to the origin of the fluid which finds its way into these organelles. If 
the origin of this fluid is associated with granules in many diverse organ- 



THE CONTRACTILE VACUOLE 411 

isms, as much of the evidence impHes, then it would be somewhat un- 1 
expected if such granules are not to be found distributed generally 
throughout the Protozoa. MacLennan called attention to the fact that 
many investigators have demonstrated a more or less solid membrane 
surrounding vacuoles in a variety of Protozoa, these demonstrations 
having been made by osmic-acid impregnation. Most of these workers, 
according to MacLennan, used the warm method of impregnation advo- 
cated by Nassonov. Hirschler showed that this method tends to produce 
overimpregnation, resulting in the production of a heavy black band or 
membrane in what is actually a granular zone. MacLennan found this 
to be true in the Ophryoscolecidae, while impregnation by the cold 
method shows this same region to be granular, a condition which can be 
seen in living material. He further calls attention to the fact that figures 
showing solid impregnation of the vacuolar walls of Chilodon and 
Dogielella, published by Nassonov, of Balantidium by Bojewa-Petms- 
chewskaja, and of the Cyclopostheiidae by Strelkow, indicate a marked 
granular roughening of the outer margin of the osmiophilic layer. He 
takes this to indicate that what has been interpreted by these authors as 
a solid membrane may, in fact, be only the result of overimpregnation 
of a granular zone. Among the Ophryoscolecidae alone MacLennan found 
various degrees of aggregation of these granules — from virtually none to 
a very pronounced aggregation — around the contractile vacuole. He fur- 
ther showed that localization of the origin of contractile vacuoles in these 
forms is correlated with the degree of aggregation of the granules. 

Of interest in this connection are the observations of Lloyd and Scarth 
(1926) on the origin of vacuoles in Spirogyra. These authors found that 
in suflficiently high concentrations even the most innocuous plasmolytes 
may by themselves cause subsidiary vacuoles to arise in the cytoplasm. It 
is not only by plasmolytes that this effect is produced however, but also 
by other more readily penetrating substances such as the narcotics, chloro- 
form, and ether, and by very low concentrations of salts. But without 
any artificial influence, similar vacuoles may form in normal cells. Their 
constant occurrence was demonstrated in the gametes during conjugation 
in Spirogyra, and their excretory function in the taking up of water from 
the central vacuole and its discharge to the exterior in typical "contrac- 
tile" fashion was proved. The authors state that these vacuoles originate 
from peculiar "lecithin-like" bodies already present in the cytoplasm. 



412 THE CONTRACTILE VACUOLE 

Scarth and Lloyd (1927) claim that the vacuolar wall arises from the 
"kinoplasm" of Strasburger. They observed a reciprocal quantitative re- 
lation between kinoplasm and mitochondria. The activity of kinoplasm 
resembles that of lecithin, which is abundant in mitochondria. On the 
basis of this resemblance, they conclude that water at least may accumu- 
late in the vacuoles without the visible interaction of any other struc- 
ture. 

The observations and opinions reported in the foregoing pages, while 
somewhat contradictory at times, point to two general conclusions con- 
cerning the origin of contractile vacuoles. First, in the great majority 
of forms, perhaps in all forms, fluid reaches the contractile vacuole 
through the fusion of small vesicles, or accessory vacuoles, with the con- 
tractile vacuole or its filling canals. The vesicles arise within what often 
appears to be the wall of the vacuole; the accessory vacuoles usually 
originate at a greater or less distance from the contractile vacuole, and 
coalesce to form the latter. Second, vesicles originate within walls of 
vacuoles which have been shown in many instances to be granular in 
nature or to be intimately associated with granules; accessory vacuoles 
have been reported by various authors as originating among granules 
which may be closely associated with the vacuole, or occasionally re- 
moved some distance from it. Thus it appears that in spite of their great 
variety of shapes and general appearances under the microscope, con- 
tractile vacuoles originate in a remarkably similar manner in all forms 
so far investigated with this problem in mind. 

In certain instances authors have reported the absence of granules in 
the vicinity of the contractile vacuole, and from this have concluded that 
granules are not concerned in the origin of vacuoles. In this connection 
it must be remembered that in certain forms, e.g., Euplotes, some of 
the Ophryoscolecidae, and apparently in others as well, the accessory 
vacuoles which ultimately give rise to the contractile vacuole originate 
at some distance from the ultimate site of the final vacuole. Also, gran- 
ules frequently are visible only after osmium or silver impregnation. 
Keeping in mind the greater vulnerability of negative evidence, one is 
justified in the thought that perhaps a reexamination of organisms for 
which the absence of granules in the vicinity of the vacuole has been re- 
ported, may reveal the presence of granules in other parts of the body, 
either scattered or in aggregates. Such scattered granules, which are known 



THE CONTRACTILE VACUOLE 413 

to exist in some amoebae, may be the site of origin of new vacuoles when 
the function of the original vacuole is disturbed by artificial means or 
removed by operation. While such granules have not been demonstrated 
to be scattered about in the cytoplasm of P. caudatum, their presence 
would explain the origin of extra vacuoles in this form, when func- 
tion of the original vacuoles is interfered with mechanically, as reported 
by Dimitrowa. The origin of new vacuoles at fission would have a similar 
explanation, since, as proposed by Dimitrowa, during fission the greater | 
abundance of metabolites would impose a necessity on the organism 
essentially similar to interference with normal function. After fission, 
when the daughter cells are smaller than the parent cell was immediately 
prior to fission, and the metabolic rate is lowered, there no longer exists 
a stimulus for the formation of extra vacuoles, and the daughter cells 
appear quite normal, with the usual number. 

The Structure of Contractile Vacuoles 

The question of the structure of the contractile vacuole and its as- 
sociated parts has occupied the attention of protozoologists for many 
years. As a result the main question has been broken up into several 
parts, each concerned with a limited phase of this main question. Is the > 
vacuole surrounded by a permanent membrane? Is its discharge to the y 
exterior through a permanent excretory pore? If there is no permanent 
pore, how may one explain the formation even of a temporary pore, and 
once formed how is it closed again? Is the vacuole a permanent structure, - 
or does it arise anew at the beginning of each new cycle? 

The dispute as to the presence or absence of a permanent membrane 
surrounding the vacuole began over a hundred years ago, and continues, 
with little to indicate that is will end within the near future. According 
to Taylor (1923), to whom we may refer for a more detailed account 
of the history of this question, the following investigators have written 
in support of the idea of a permanent membrane: Ehrenberg, Siebold, 
Claparede, Lachmann, Degen, and Stempell. Those who believe that 
the vacuole possesses no permanent wall are: Dujardin, Meyen, Stein, 
Wrzesniowski, Perty, Schmidt, Zenker, Maupas, Rhumbler, Biitschli, Lan- 
kester, and Khainsky. Taylor himself holds this view, at least for Eu- 
plotes. Without reflecting unfavorably in any way on the researches of 
those who worked on this subject prior to 1900, or possibly as late as 



414 THE CONTRACTILE VACUOLE 

1920, one must admit that only limited importance can be attached to 
their opinions. Unquestionably most of these investigators were careful 
observers, and expressed opinions only after due consideration of all the 
factors which they were able to recognize. But the microscope of today 
is a far different instrument from that of a hundred years ago, or even 
fifty years ago; and chemical procedures, particularly those dealing with 
colloids, have undergone extensive development. However highly one 
may regard this or that early investigator, the fact remains that none 
could have been better than the tools with which he worked, and ad- 
mittedly the tools were poor. Consequently, the author maintains that 
it is neither unkind nor unappreciative to propose that these various early 
opinions be considered mainly as of historical interest, and of little 
worth in settling the question as to the presence or absence of permanent 
membranes, or of any kind of membrane for that matter, around con- 
tractile vacuoles. The employment of the best of modern instruments 
and techniques leaves the question in an unsatisfactory state. 

Before presenting the more recent evidence concerning this question 
of membranes, perhaps it will not be unwise to present briefly the more 
fundamental question of what constitutes a membrane. Most textbooks 
either avoid the issue more or less completely or describe the structure 
and properties of the artificial membranes so often used in the laboratory 
for experimental purposes. Although reliable information concerning 
living membranes is scant, there is sufficient evidence to justify the divi- 
sion of membranes into two types: morphological membranes and physi- 
ological membranes. Morphological membranes are permanent struc- 
tures which are frequently visible in living material viewed through the 
microscope, and usually may be demonstrated more or less clearly by 
suitable staining techniques. Apparently they consist mainly of a reticu- 
lum, or framework, which is described by some authors as being com- 
posed largely of protein. Such membranes are usually thought to possess 
an appreciable amount of rigidity, and to serve primarily as supporting 
structures. Free permeability in both directions is usually assigned to 
them. 

Physiological membranes are entirely different from morphological 
membranes in many important respects. Usually they are considered to 
be so thin as to be invisible even with the highest magnification. They 
may possess a certain degree of rigidity, but probably much less than 



THE CONTRACTILE VACUOLE 415 

morphological membranes with which they are often associated in liv- 
ing material. Semipermeability, or more properly selective permeability, 
is a property of all living physiological membranes. Colloid-chemists, 
as well as many physiologists, are agreed that a physiological membrane 
is simply a phase boundary, an interface between two different fluids. 
In order that such phase boundary may be more or less permanent, it 
is necessary that the two phases be only slightly miscible at most — the 
more complete the immiscibility the more nearly perfect and permanent 
the membrane. A very wide variety of molecules show polar phenomena; 
that is, the two ends of the molecules are electrically and chemically 
different. This results in orientation of molecules with respect to one 
another and to various other molecules, in much the same manner that 
a compass needle becomes oriented with respect to the magnetic poles of 
the earth. This phenomenon of orientation is associated with organic 
acids, alcohols, aldehydes, lipoids, fats, proteins, and many other so- 
called "physiological" compounds. Thus at the interface of the two- 
phase system, oil-water, the oil molecules (glycerol-esters of fatty acids) 
become oriented in such a manner that the hydrocarbon ends of the 
molecules project into the oil phase, whereas the glycerol ends project 
into the water. Fat molecules undergo much more nearly perfect orienta- 
tion than water molecules, although with the latter there appears to be 
a certain degree of orientation. Such an aggregation and packing to- 
gether of oriented polar molecules at an interface represents a physio- 
logical membrane. Since protoplasm contains a variety of polar molecules, 
the membrane formed between protoplasm and water is composed of 
various types of molecules apparently arranged in the form of a mosaic. 
The thickness of such a membrane has not been definitely established. 
Some authors maintain that it is only a single molecule thick, or at most 
only one or two milli-micra thick, but at least one author (Peters, see 
Clark, 1933, p. 40) has advanced a theory according to which the cell 
is composed of a three-dimensional protein mosaic, with the molecules 
in the interior of the cell oriented on the surface film. Since the interior 
of many cells is known to be fluid, the structure must be regarded as an 
orientation rather than as an anatomical skeleton. This theory of Peters's 
agrees fairly well with certain evidence concerning the action of drugs 
on cells. Without entering into the question as to how far orientation 
extends beneath the surface layer, suffice it to say that it is well established 



416 THE CONTRACTILE VACUOLE 

that orientation of surface molecules occurs at the interface between two 
different phases (provided, of course, that at least one phase contains 
polar molecules), whether the system is composed of oil and water or 
protoplasm and water. The converse of this is equally true; since the 
very existence of this oriented layer depends on the presence of tivo 
different phases, the removal of one phase necessarily results in disintegra- 
tion of the membrane. Whether or not this surface layer of oriented 
molecules actually comprises the true physiological membrane may be 
subject to debate, but the importance of such a membrane is obvious, 
since it not only separates the organism from its surroundings but at 
the same time provides the only means of communication between the 
interior and the exterior of the cell. 

If one accepts the idea of a physiological membrane as a layer of 
oriented molecules at a phase boundary, as outlined briefly above, then 
it necessarily follows that any cell vacuole which contains a fluid differ- 
ent from cytoplasm must be surrounded by such a membrane. In the 
light of the information available at the present time, membranes around 
protozoan contractile vacuoles probably should be considered as tem- 
porary; although it is not inconceivable that in some forms the new vac- 
uole may be formed with such rapidity, in the midst of oriented mole- 
cules remaining after systole, that dispersion of the membrane does not 
have time to occur before the second phase is present again. If such condi- 
tion obtains, then the membrane may be considered as having a greater 
or less degree of permanence. There are numerous references to such 
physiological membranes in the literature on contractile vacuoles, so it 
appears that the idea has gained rather wide acceptance. The controversy is 
not so much concerned with such membranes as with the presence or 
absence of morphological, and hence permanent, membranes. 

Before leaving the subject of physiological membranes, there are sev- 
eral phenomena which may be discussed profitably with this concept in 
mind. Repeatedly authors speak of the coalescence of accessory vacuoles 
to form contractile vacuoles. Taylor (1923) refers to this, and considers 
coalescence to be due to a reversion of the gel state of the surrounding 
film to the sol state. He further states that vacuoles are surrounded by 
highly viscous boundaries of endoplasm, and that the consistency of the 
papilla pulsatoria strikingly resembles that of the endoplasmic boundaries. 
Without raising the question as to whether or not coalescence of accessory 



THE CONTRACTILE VACUOLE 4l7 

vacuoles and the simultaneous rupture of vacuole wall and papilla pul- 
satoria represent sol-gel reversibility, it is obvious that exactly these 
phenomena must be anticipated, on the basis of the concept of physiologi- 
cal membranes such as described above. When two accessory vacuoles lie 
touching each other, the cytoplasmic phase of the two-phase system is 
pushed aside, at the same time removing the basic forces on which the 
presence of these membranes depends. In the absence of these forces, 
the membranes disintegrate at the site of contact, and the two vacuoles 
fuse into one. Likewise, when the filled vacuole comes in contact with 
the papilla pulsatoria, one phase (the cytoplasmic phase again) is pushed 
aside, the membranes disintegrate at the site of contact, and the contents 
of the vacuole are discharged to the exterior. After discharge the two- 
phase system is again established, since there is cytoplasm on one side of 
the pore (within the organism) and water on the other (outside the 
organism), whereas prior to discharge there was water on the outside as 
well as a fluid composed chiefly of water within the vacuole. The papilla 
is formed in this manner from the vacuole wall, which readily accounts 
for the similarity noted by Taylor. 

Other phenomena which can be explained in like manner by the pres- 
ence of physiological membranes are easy to find in the Protozoa. The 
ingestion of food by Amoeba is essentially the result of fusion of the 
walls of the organism after they have been extruded around the food 
particle in such a manner as completely to enclose it. The two-phase 
system exists as long as there is water on one side of the cell membrane 
and cytoplasm on the other; but when the engulfing process is complete 
and cell membrane is in contact with cell membrane with no water 
separating the two portions, the membrane disintegrates at the site of 
contact, and continuity of cell structures as well as of the vacuole mem- 
brane is established. The food-vacuole membrane persists as long as 
there is water within to maintain the two-phase system. In a similar man- 
ner one may explain the readiness with which an amputated fragment 
of an amoeba unites with the parent body when the two portions come 
together, although it has no bearing on the fact that a fragment from a 
diverse strain is refused. It has also been observed that occasionally an 
amoeba attempts to engulf a relatively large organism, such as P. cauda- 
tuni, but is unable to accomplish this completely. The Paramechim is 
squeezed in two, apparently, with half inside the amoeba and half out- 



418 THE CONTRACTILE VACUOLE 

side. Calculations have been made of the physical force necessarily ex- 
erted by the amoeba to accomplish this, but they have not taken into 
account some of the properties of physiological membranes, such as spon- 
taneous disintegration when membrane comes into direct contact with 
membrane. The adherence of conjugating organisms may be dependent, 
likewise, on these same properties of membranes. While it is interesting 
to speculate on such matters, it must be admitted that these remarks on 
feeding amoebae are purely speculative, with little other than superficial 
observation to support them. 

Concerning the presence of a permanent (morphological) membrane 
surrounding the contractile vacuole, there are diverse opinions. These 
diverse opinions apply not only to different species but even to the same 
species. Howland (1924a) found that the contributory globules as well 
as the vacuole may be removed from the organism to the surrounding 
water, where they retain their identity for an indefinite period of time. 
This may be taken to indicate a considerable degree of permanence of the 
vacuole wall, such as would be possessed by a morphological membrane, 
although Howland is of the opinion that these vacuole membranes are 
temporary. A temporary physiological membrane, formed of oriented 
molecules in a compact layer, may be expected to retain its identity for 
an appreciable period of time before dispersion of the molecules occurs, 
but it hardly seems probable that this "appreciable period of time" can 
be more than a few minutes. Day (1927) expresses the opinion that the 
vacuole wall in A. proteus is a "condensation membrane," or gel, disap- 
pearing with each contraction. By "condensation membrane" Day very 
likely implies such a structure as has been described above as a physiologi- 
cal membrane, so that the two terms may be taken as synonymous. Con- 
cerning A. proteus, Mast (1938, p. 307) states: "At the surface of the 
contractile vacuole under the layer of substance containing the beta gran- 
ules, there is a layer or membrane about 0.5 micron thick which is opti- 
cally well differentiated from the adjoining substance on either surface, 
for under favorable conditions a line indicating an interface can be clearly 
seen at both these surfaces." Whether this membrane is a permanent 
structure or is formed anew with each successive vacuole was not sug- 
gested by Mast. After an examination of fixed, stained, and sectioned 
material, Haye (1930) concluded that vacuole membranes are lacking 
in Amoeba, although it is probable that this author referred to morpho- 



THE CONTRACTILE VACUOLE 419 

logical and not to physiological membranes, since these latter cannot 
be demonstrated in this manner. 

Among the flagellates, Nassonov (1924) found an osmiophilic mem- 
brane surrounding the vacuole in Chilomonas Paramecium. Haye ( 1930) 
could distinguish no vacuole wall in the Euglenoidina, but in many other 
flagellates examined by him distinct walls were visible in stained ma- 
terial. 

Among the ciliates, morphological membranes are reported in a great 
variety of organisms by many investigators. Nassonov (1924) reports 
osmiophilic walls for the vacuoles in Paramecium caudatum, Lionotus 
joUum, Nassula laterita, Cam panella umhellaria, Epistylis gallea, Zootham- 
nium arbuscula, and Vorticella sp. Faure-Fremiet (1925) confirmed 
the findings of Nassonov, using several species of Vorticella, in which 
osmiophilic walls were observed, even after collapse of the vacuole. 
Young (1924) concludes from studies on P. caudatum stained with iron 
hematoxylin that the vacuole system is a permanent and continuous struc- 
ture. King (1928) arrived at essentially the same conclusion concerning 
the vesicle-fed system of P. trichium. Wenrich (1926) observed definite 
vacuole walls in P. trichium stained with Mayer's hemalum or Heiden- 
hain's iron-alum-hematoxylin. Concerning this Wenrich states (p. 89) : 

It was somewhat surprising to find how distinctly the vacuolar walls showed 
in fixed and stained specimens. The relative thickness of the wall is note- 
worthy and it usually appears to be laminated. In sectioned material the walls 
contained strands of more or less intensely staining material, suggesting 
the presence of contractile fibers. 

Von Gelei observed osmiophilic walls in P. caudatum (1925, 1928) 
and in Spathidium giganteum (1935). Haye (1930) found thin vacuole 
walls in the following forms: Blepharisma undulans, Lionotus fasciola, 
Ophrydium versatile, Stenlor polymorphus, Spirostomum ainhiguum, 
Balantidium entozoon, and Isotricha prostoma. Thick walls were ob- 
served in Campanella, Chilodon, Dogielella, Paramecium, and the Oph- 
ryoscolecidae. As previously mentioned, Nassonov (1925) examined 
Chilodon and Dogielella after osmium impregnation and concluded 
that the vacuoles possess no membranes, but lie directly in the cytoplasm. 
In these latter organisms, structures considered by Nassonov to be the 
Golgi apparatus surround the vacuole in such a manner that they may be 
mistaken for vacuole walls in certain preparations. 



420 THE CONTRACTILE VACUOLE 

The presence of definite vacuole walls or morphological membranes 
around the vacuoles of ciliates is not accepted by all authors. Thus, Taylor 
(1923) believes the vacuole in Enplotes to disappear completely at sys- 
tole, and to be replaced by an entirely new structure. If a morphological 
membrane were present, this could hardly obtain, although Taylor was 
able to distinguish a "highly viscous boundary" of endoplasm surround- 
ing the vacuole. Moore (1931) was unable to cause osmication of the 
vacuole wall in Blepharisma undid ans, using the technique of Nassonov, 
and from this she concludes that the vacuole lacks a permanent wall. 
This opinion was again expressed (1934) after further observation. 
The findings of Moore are in opposition to those of Haye, who re- 
ported thin vacuole walls in the same species. Day (1930) concludes 
from his observations on Paramecium caudatum, Spirostomum amhi- 
guum, and 5". teres that vacuoles in these forms are temporary structur