Materials and Methods in the Study of Protozoa [Reprint 2020 ed.] 9780520348141

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MATERIALS AND METHODS IN THE STUDY OF PROTOZOA

BY HAROLD KIRBY Professor of Zodlogy, University of California, Berkeley

COPYRIGHT, 1950, BY THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

Price: $2.50

UNIVERSITY OF CALIFORNIA PRESS BERKELEY AND LOS ANGELES CALIFORNIA • *«

CAMBRIDGE UNIVERSITY PRESS LONDON, ENGLAND

PRINTED BY OFFSET IN THE UNITED STATES OF AMERICA

" I n pursuing our r e s e a r c h e s we have become practically convinced of the importance of what we have theoretically assumed - - the absolute necessity for prolonged and patient examination of the same f o r m s . Two observers, independently of each other, examining the same monad, if their inquiries were not sufficiently prolonged, might, with the utmost truthfulness of interpretation, a s s e r t opposite modes of development. Competent optical means, careful i n t e r p r e tation, close observation, and time are alone capable of solving the p r o b l e m . " - - Dallinger and Drysdale, 1874

PREFACE

Information given in this manual was originally compiled for use in instruction at the University of California, in order to have in readily a c cessible f o r m some schedules of procedure. Among the data are techniques that are followed routinely in laboratory operations. But the compilation has been extended beyond provision of directions for routine procedure for a laboratory course. It deals with methods that a r e of value in providing and preparing materials for laboratory instruction and investigation, and with some techniques that may be useful in meeting special requirements, There is, however, no pretense to completeness in treatment of protozoological methods or in presenting and evaluating advanced and refined techniques. The ways of collecting, cultivating, preparing, and studying p a r ticular protozoa must be adapted to each situation, and the most suitable methods must be learned by individual experience. There must always be a beginning, and even an inadequate survey of established procedures is useful in laying a foundation and organizing a framework within which to work. The microscope reveals a world of living things and there a r e many ways of investigating that world. One person may feel that knowing the protozoa holds his interest ho less than knowing the birds or t r e e s or insects holds that of many persons. He may be content with collection and observation of living organisms. Another will study the relation of protozoa to one another and to the organic and physical environment. Another, again, will wish to p r e s e r v e specimens for c o m p a r a tive studies, and to explore the details of cytology. The manifold types of life history, the problems of sexual differentiation and inheritance, the physiological activities, and the nutrition of protozoa a r e other m a t t e r s f o r the concern of biologists. The student who continues to work with these organisms ultimately chooses a r e s t r i c t e d phase of investigation. It may be a study of some aspects of the biology of protozoa, it may be a practical phase such a s the diagnosis of protozoan infections of economic and medical importance, or the use of F o r a m i n i f e r a and Radiolaria in geological exploration. However it may be, he uses one set or another of standard techniques. The techniques dealt with here a r e mostly ones for securing, maintaining, and making preparations of protozoa for observation. One of the useful features of this book should be the presence of blank pages opposite the printed pages. On them can be entered the corrections, additions, and modifications that will undoubtedly be necessary. Acknowledgment is humbly and gratefully made to the authors and publ i s h e r s of many books and articles f r o m which information about methods has been obtained; and to the persons, associated with this author at one time or a n o t h e r , f r o m whom he has learned the results of their experience. HAROLD KIRBY

Berkeley, California June, 1950

CONTENTS Section

Page

I. COLLECTION AND CULTIVATION METHODS FOR FREE-LIVING PROTOZOA GENERAL COLLECTION METHODS KINDS OF CULTURES CULTURE VESSELS ISOLATION TECHNIQUE MATERIALS AND PROCEDURE Chemical formulas of inorganic compounds in culture media Agar media

1 1 1 1 2 3 3 3

Allen solution, for marine forms Amoeba medium

4 4

Amoebozoa

4

Arcella

5

Bacteria-eating protozoa Barker medium for marine dinoflagellates Benecke solution

5 5 5

Bodonid flagellates Cereal medium Chilomonas

5 5 5

Chloromonad flagellates Chrysomonad flagellates Ciliata Citric acid Colpodid cillâtes Commercial fertilizer Coprophilic protozoa

6 6 6 6 6 6 6

Dldinium nasutum

7

Dimastigamoeba gruberi Dinoflagellates Dunaliella Egg yolk medium Euglena Euglenoid flagellates Feeding of cultures Flagellata Flagellates of brine . . Flour-Hay medium Foraminifera Hahnert solution Hay infusion Heliozoa Hypotricha Klebs solution Knop solution Lackey wheat medium Lefèvre medium Lettuce infusion Light Meat extract Molisch solution Moore solution Mycetozoa Paramecium Peat extract Pelomyxa Peters medium

.

7 7 7 7 7 8 8 8 8 9 9 9 9 10 10 10 10 11 11 11 11 11 11 11 11 12 12 12 13

Physiological balanced solution

13

Phytomonad flagellates

13 [vii]

viil

CONTENTS Proteomyxid rhizopods Radiolaria Soil extract Soil protozoa Spirostomum Stentor Suctorla Synthetic sea water Synthetic spring water Tetrahymena geleii Vol vox Vorticella Wheat infusion Yeast medium for excystation Zumstein medium

Page 13 13 14 14 14 14 14 14 14 15 15 15 15 15 15

REFERENCES ON COLLECTION AND CULTIVATION OP FREE-LIVING PROTOZOA

15

ADDENDUM —

17

Soil-cheese medium

II. COLLECTION AND CULTIVATION METHODS FOR SYMBIOTIC PROTOZOA MATERIALS AND PROCEDURE Agar-Serum media Amoebosporidia Amoebozoa Blood-Agar medium Blood-Ringer medium Blood-Serum medium Ciliata Ciliate cultivation Egg slants Egg Yolk infusion medium Fecal extract medium Flagellata . . Fluid media Histomonas meleagridis Host examination Intestinal flagellates and amoebae Isolation from bacteria Liquid media to cover slants Liver infusion agar . Locke solution Lophomonas blattarum Opalinid ciliates Preservation by low temperature freezing Ringer solution Serum slants Sporozoa Starch . . Trichomonad flagellates Trichomonas vaginalis Tritrichomonas foetus Trypanosomatidae REFERENCES ON COLLECTION AND CULTIVATION OF SYMBIOTIC PROTOZOA III. TECHNICAL METHODS OF STUDY AND PRESERVATION SCHEDULES AND PROCEDURE Aceto-carmine Adherence to slides and cover glasses Amoebozoa Best's carmine Bleaching Blood examination

18 18 18 18 18 19 19 19 19 20 20 20 21 21 22 22 23 23 24 24 24 24 24 25 25 25 25 25 27 27 27 27 28 28 31 31 31 31 31 32 32 32

CONTENTS Blood films Borrel stain Buffer solutions Camera lucida . . Carmine staining Cellulose Chlor-zinc-iodine Chondriosomes Cilia staining in permanent preparations Cilia and flagella staining in water Cleaning slides and glassware Clearing Concentration and cleaning of protozoa Concentration of cysts Copper sulfate Dinoflagellate plates Drawing . Enumeration of net plankton catch Enumeration of organisms Feulgen nucleal reaction Fixation fluids Benoit fluid Bouin fluid Brasil-Duboscq modification of Bouin fluid Carnoy fluid Champy fluid Da Fano fixative Flemming fluid Formalin Gelei Sublimate-dichromate-alum fixative Heidenhain Susa fixative Hermann fluid Hirschler fluid Hollande cupric picro-formol Osmic acid Perenyi fluid Petrunkevitch fluid . Schaudinn fluid Sublimate acetic Worcester fluid Yocum fluid Zenker fluid Fixation fluid mixtures from stock solutions Fixation time and temperature Flagellata Foraminifera Formalin Giemsa staining Staining dry blood films Staining wet-fixed preparations Glycerine jelly Glychrogel Glycogen Golgi bodies Gram solution Gregarines Haematoxylin staining Iron haematoxylin staining Iron haematein staining Counterstaining Alum haematoxylin staining Tungstic haematoxylin staining Hydrogen-ion concentration, determination of Illumination Immobilization Iodine

ix

.

. . . .

32 33 33 33 33 34 34 34 35 35 36 36 36 36 37 37 37 37 38 39 39 39 39 39 39 40 40 40 40 40 40 40 40 40 40 4l 4l 4l 4l 4l 4l 4l 4l 42 42 43 43 43 44 44 44 45 45 45 45 45 46 46 47 47 47 48 48 49 49 49

x

CONTENTS J.S.B. stain for blood parasites Laboratory procedure in microscopy Leishman stain Lipids Loeffler stain for flagella Lugol solution M a n n methyl blue eosin stain Mayer albumen fixative Measurement Mechanical stage readings Methyl g r e e n acetic Microincineration Microscopy (See Laboratory procedure in) Mounting m e d i a Nuclear staining in temporary preparations Paramecium and similar ciliates Protein reactions Rapid preparation of permanent stained mounts Relief staining Sealing preparations Sectioning Shape preservation Silver impregnation Sporozoa and Amoebosporidia Sporulation of obcysts Spore filament discharge Starch Termite flagellates Trichomonad flagellates Vital and supravital staining Volutin W r i g h t stain

*

Page 50 50 50 51 51 51 51 51 52 52 52 52 52 52 53 53 55 55 56 56 56 56 57 59 59 59 59 59 59 60 60 60

REFERENCES ON TECHNICAL METHODS

60

ADDENDUM —

63

INDEX

R e g a u d haematoxylin

65

I. COLLECTION AND CULTIVATION METHODS FOR FREE-LIVING PROTOZOA GENERAL COLLECTION METHODS

the l o w e r side w h e n the slide is h o r i z o n t a l . I m m e r s e d s l i d e s m a y b e u s e d f o r c o l l e c t i o n of protozoa in fresh water or in sea water. VariP l a n k t o n forms are c o l l e c t e d b y m e a n s of a ous r h i z o p o d s , a t t a c h e d p e r i t r i c h s , a n d t h i g m o n e t of the f i n e s t g r a d e of b o l t i n g silk. In tactic hypotrichs m a y be captured, among other l a k e s , the net c a n b e s t b e u s e d from a boat. forms. T h e f o r m s c o l l e c t e d s h o u l d be b r o u g h t to the l a b o r a t o r y In b o t t l e s (thermos b o t t l e s if the Marine plankton protozoa m a y be collected a i r t e m p e r a t u r e is high) and e x a m i n e d as s o o n as b y f i l t e r i n g o r c e n t r i f u g i n g s a m p l e s of s e a possible. w a t e r , or f o r the l a r g e r s p e c i e s a f i n e p l a n k t o n net m a y b e used. L a c k e y (1936) s e c u r e d a v a r i P l a n k t o n forms m a y b e p r e s e r v e d b y a d d i n g e t y of b o t t o m - d w e l l i n g m a r i n e p r o t o z o a o n S y r a f o r m a l i n to the w a t e r to m a k e about a 5$ s o l u c u s e d i s h e s e n c l o s e d i n w i r e c a g e s to e x c l u d e tion; or t h e y m a y b e c o n c e n t r a t e d , f i x e d a n d l a r g e r o r g a n i s m s a n d s u s p e n d e d f r o m a f l o a t at stained b y appropriate techniques. W o o d s H o l e . W i t h i n 2 h h o u r s m a n y t h o u s a n d s of The smallest forms will not be taken b y a i n d i v i d u a l s of m a n y s p e c i e s of a m o e b a e , f l a g e l p l a n k t o n net. To s e c u r e them the w a t e r m u s t b e l a t e s , c i l i a t e s , a n d one species of s u c t o r i a n f i l t e r e d or c e n t r i f u g e d . S a m p l e s of w a t e r f r o m l a k e s , ponds, streams, h a d a c c u m u l a t e d o n a s q u a r e c e n t i m e t e r of a n i m Enrichment cultures m a y be prew a t e r t r o u g h s , and o t h e r places m a y b e c o l l e c t e d m e r s e d dish. p a r e d b y a d d i n g to f i l t e r e d s e a w a t e r n u t r i e n t i n jars. M e r e l y d i p p i n g into the w a t e r u s u a l l y m a t e r i a l s a n d the sample to b e s t u d i e d . will not yield very much. W a t e r samples s h o u l d g e n e r a l l y b e t a k e n w h e r e there is plant g r o w t h or organic debris. The w a t e r to fill the j a r K I N D S OP C U L T U R E S m a y b e in part e x p r e s s e d f r o m the plants, and some of the p l a n t m a t e r i a l s h o u l d b e t a k e n along, A raw s a m p l e , o b t a i n e d f r o m the f i e l d , c o n b u t the jar s h o u l d not b e f i l l e d w i t h it. The c o l l e c t i o n m a y also i n c l u d e samples of a n y s l i m y tains a m i x t u r e of o r g a n i s m s w h i c h are n o t u n d e r g r o w t h s that are p r e s e n t o n the b o t t o m or o n o b - c o n t r o l , a n d as the c u l t u r e a g e s the p o p u l a t i o n m a y rapidly change in character. jects i n the w a t e r , d e a d l e a v e s , some s a n d f r o m the b o t t o m , pieces of sticks f r o m the w a t e r , a n d A n i m p u r e s p e c i e s c u l t u r e of a p r o t o z o a n s u r f a c e scum. c o n t a i n s one species as the d o m i n a n t f o r m , . a n d b y suitable m e t h o d s of f e e d i n g a n d t r a n s f e r it A f t e r r e a c h i n g the l a b o r a t o r y , the c o l l e c can be maintained. A l o n g w i t h the s p e c i e s are t i o n s s h o u l d b e put out i n c r y s t a l l i z i n g d i s h e s o r r e f r i g e r a t o r jars. The bottom may be covered unknown bacterial populations and other organb y s a n d o r soil a n d some plant m a t e r i a l included. isms. M o s t p r o t o z o a f o r g e n e r a l l a b o r a t o r y u s e are m a i n t a i n e d in c u l t u r e s of this sort. I n a n a q u a r i u m of t h i s sort the p r o t o z o a o r i g i n a l l y a c t i v e at the site w h e r e the c o l l e c t i o n A g r e a t e r d e g r e e of c o n t r o l is o b t a i n e d w a s m a d e m a y live for some time; t h e y u s u a l l y w h e n a species is k e p t f r e e of o t h e r o r g a n i s m s w i l l not b e c o m e e s p e c i a l l y abundant. If m o r e e x c e p t those that are a d d e d f o r f o o d . This o r g a n i c m a t e r i a l is a d d e d , there will b e a n inmethod requires more refinement in original isoc r e a s e i n n u m b e r s of some f o r m s , b u t others will l a t i o n a n d m a i n t e n a n c e . perish. In o r d e r to o b t a i n large p o p u l a t i o n s , A pure c u l t u r e is one i n w h i c h the p r o t o c u l t i v a t i o n m e t h o d s are u s e d . z o a n is the o n l y o r g a n i s m . T h e c u l t u r e is m a i n t a i n e d b a c t e r i a - f r e e , a n d the m e d i u m is a r i c h M a n y p r o t o z o a m a y be c a p t u r e d on s l i d e s or o r g a n i c one w h i c h serves f o r n u t r i t i o n of the c o v e r g l a s s e s i m m e r s e d in w a t e r . One m e t h o d of These media usually cannot be used o b t a i n i n g some of them is to f l o a t cover g l a s s e s p r o t o z o a n . o n the s u r f a c e ; this m e t h o d c a n b e u s e d s u c c e s s - w h e n m u l t i p l y i n g b a c t e r i a are p r e s e n t . f u l l y o n l y in i n d o o r c o n t a i n e r s w h e r e the w a t e r is q u i e t . S l i d e s i m m e r s e d in the w a t e r in a q u a r i a , or i n the n a t u r a l e n v i r o n m e n t , w i l l CULTURE VESSELS p r o v i d e m a n y forms for o b s e r v a t i o n or f o r m a k i n g preparations. Slides c a n b e h e l d in g r o o v e s in C u l t u r e s m a y be k e p t i n c o n t a i n e r s of v a r i a piece of w o o d , w h i c h is f a s t e n e d to a s u p p o r t ous s o r t s , a c c o r d i n g to the r e q u i r e m e n t s a n d to u n d e r w a t e r o r to a stake i n s e r t e d in the bottom. the d e g r e e of control that is u s e d . F a u r ^ - P r e m i e t (1931) r e c o m m e n d e d s u p p o r t i n g t h e m V e s s e l s of o r d i n a r y g l a s s , s u c h as r e f r i g i n g r o o v e s in a w o o d e n frame s i m i l a r to a slide erator dishes, drinking glasses, and jars, m a y box without top or bottom. The slides are s e p a - be a d e q u a t e f o r m a i n t a i n i n g m a n y g e n e r a l c u l r a t e d b y a b o u t 1 c e n t i m e t e r , and are h e l d i n the t u r e s . The glass m a y have unsuitable qualities, f r a m e b y strips of wood. The f r a m e m a y b e a n d m o r e r e f i n e d g l a s s of t h e type u s e d i n m a k f l o a t e d i n the w a t e r , o r f i x e d v e r t i c a l l y . Ating l a b o r a t o r y a p p a r a t u s is o f t e n b e t t e r . t a c h e d o r g a n i s m s o c c u r in the b e s t c o n d i t i o n o n F o r c u l t u r e s w i t h small n u m b e r s of t h e [1]

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA protozoa and for frequent transfer depression slides are used. • These may be kept in a closed dish in a saturated atmosphere. Syracuse watch glasses are useful for larger populations and for cultures following isolation when mass cultures are being built up. Small Petri dishes may also be used. Finger bowls (laboratory type) are useful for maintenance of mass cultures. They can be stacked one on another to provide covering and save space. Cultures of amoebae and other forms may be kept in such containers, and observations can be made directly under a stereoscopic microscope . When more fluid is used, moist chambers of larger size are suitable containers. A greater volume of fluid is an advantage in cultures that are to be maintained for a long time with repeated feeding. Erlenmeyer flasks are good for maintenance of mass cultures of many forms, but not for bottom-dwelling types such as rhizopods, which are best kept in moist chambers without great depth of water. The liter-size flask is useful for mass cultures of such protozoa as Euglena, Paramecium, and other free-swimming ciliates. The flasks can readily be sterilized while they are stoppered with cotton, and evaporation is hindered, especially if a small beaker is inverted over the top. Samples can be taken and transfers made simply by pouring from the flask. The flask should be filled to not more than half its height. Cotton-stoppered test tubes can be used for cultures of various sorts, and may be employed in the maintenance of pure cultures. Agar culture media may be made in slants in test tubes or placed in Petri dishes. Unless crude mixed cultures are all that is desired, the glassware should be well cleaned and sterilized before use. It should not have been cleaned with sulfuric acid and dichromate cleaning solution. Wichterman (19^9) recommended immersing in 10$ nitric acid solution. It should never have been in contact with formalin, fixatives, or poisons. Even formalin fumes in the same room are detrimental to cultures . ISOLATION TECHNIQUE It is desirable for many purposes to begin cultures from single specimens of protozoa. When this is not necessary, but it is desired to maintain only the one species, cultures may be started from several specimens together; often the group does better than a single individual would. Wild cultures brought into the laboratory may be examined under a stereoscopic microscope for the desired forms. These are then picked out with a pipette drawn out to a small end. The glass tubing used to make the pipette should not be too thin. Mouth pipettes are frequently used; a long rubber tube is held in the mouth

2

and aspiration regulated by means of the breath. A rubber nipple on the pipette, regulated by pressure of the fingers, is often satisfactory enough. Transfer through a series of two or three watch glasses may facilitate freeing the forms desired of other species of protozoa. In beginning a culture, use of a depression slide with as large an amount of culture medium as possible may be advantageous. Acclimatization is often necessary; direct change from the original fluid to the culture medium may be unfavorable. The culture medium to be used may be diluted with the original fluid and the volume of fluid increased with higher proportions of culture medium as the organisms multiply. Certain protozoa may serve as food for certain other protozoa. To maintain the latter, the food forms are cultivated separately and given as required. It may be desirable to free the protozoa of bacteria, in order to keep them in pure culture or feed them on known bacteria. Some methods of doing this are described by Trager (1937), Kidder (19^1), Hetherington (I93^a), Phelps (193^), and Claff (19^0). In the migration method described by Trager, pipettes 14 inches long,, with l/4 inch bore at one end and tapering at the other end are used. The large end is plugged with cotton and a rubber tube attached. The apparatus is sterilized and sterile water drawn up to within 2 inches of the top. 2 cc. from a rich culture of the protozoan are then sucked in, and the tapered end is sealed by heat. The apparatus is kept with the larger end up, and protozoa may migrate to the top in 5 to 30 minutes, leaving most or all of the bacteria behind. The procedure may be repeated once or twice, taking into a fresh sterile pipette, with sterile water to within 2 inches of the top, about 2 inches of fluid from the top of the previous pipette. When the protozoa that are being freed of bacteria are positively geotropic, the sample from the culture is placed on top of the column of water in the first pipette. After migration has taken place the sealed lower end is cut off and fluid from it used for inoculation, or if not yet bacteria-free, is added to the top of the column of water in a second pipette. Hetherington(l93^a) sterilized ciliates by repeated migration in successive transfers in watch glasses. Seven watch glasses are enclosed in Petri dishes. Transfer pipettes are cottonstoppered at the bulb end. Glassware and pipettes are sterilized. Sterile medium is placed in each of the series of watch glasses. A concentrated mass of the protozoa is placed at the left margin of the fluid of the first dish, and after migration to the opposite side (observed by a stereoscopic microscope) some of the protozoa are removed to the left margin of the fluid in the second dish. Similar migrations are allowed to take place in all the dishes of the series in succession, washing the organisms in the third and fifth dishes by leaving them in

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA each for three hours. After the final migration, the organisms are introduced directly into the culture medium to be used. The apparatus devised by Claff (1940) consists of a series of 6 small flasks, the top of each joined by tubing to the bottom of the next, the first connected to a large flask reservoir from which fluid is received, the tube from the top of the last flask opening into a test tube. The protozoa are introduced by a hypodermic syringe through a vaccine port near the bottom of the first flask. They migrate to the top and are washed by release of fluid from the reservoir into the bottom of the second flask; as the migration and dilution process continues they may be freed of bacteria. A critical review of methods used in sterilizing Paramecium was given by Wichterman (19^9)> He pointed out that it is necessary to provide against contamination of cultures by spores of bacteria carried through the washing and migration processes within the body. When the ciliates remain for several hours in sterile water, it is likely that the spores will have been eliminated from food vacuoles. Seaman (19^7) reported sterilization of ciliates (Colpidium campylum) by use of penicillin. Ciliates from a wild culture were concentrated, washed, and placed in culture medium (3$ proteose-peptone) with 5000 units penicillin per cc. for 12 hours. It is suggested that comparable methods may be satisfactory in obtaining sterile cultures of other protozoa, but for some of them a different concentration of penicillin and different times of exposure may be necessary.

Potassium nitrate Potassium phosphate, monobasic Potassium phosphate, dibasic Sodium bicarbonate Sodium bromide Sodium carbonate Sodium chloride Sodium hydroxide Sodium phosphate, monobasic Sodium phosphate, dibasic Sodium sulfate Sodium sulfide, crystal.

3 KNO3

KH2PO4 K 2 HP04 NaHCOo NaBr Na 2 C0 3 NaCl NaOH NaH2P0^.H20 Na 2 HP04 Na 2 S0^ Na 2 S.9H 2 0

AGAR MEDIA Agar media may be used for chrysomonads, autotrophic cryptomonads, the simpler phytomonads, some euglenoids, cercomonads, bodonids, Mycetozoa, small amoebae, small shelled rhizopods, and some small ciliates. Agar in the amount of 1$ to 2$, or less, is dissolved in the heated fluid. The solution is poured into Petri dishes, or into test tubes in which it is allowed to harden in a slant.

For freshwater forms: Knop agar. 1$ agar in 0.05$ KNOP SOLUTION. Alkaline Knop agar. 2$ agar in 0.05$ Knop solution made alkaline to litmus with 10$ sodium carbonate solution. Bouillon agar. (See under MEAT EXTRACT.) Beneeke agar. 1$ agar in 0.05$ BENECKE SOLUTION. Meat extract agar. 2$ agar in 1000 cc. spring water, + 0.3 -- 0.5 grams meat extract. Musgrave and Clegg agar medium. Agar 2 . 5 MATERIALS AND PROCEDURE grams, sodium chloride 0.05 grams, (For convenience in finding, the topics are beef extract 0.05 grams, normal sodium arranged in alphabetic order. Cross references hydroxide solution 2 cc., water 100 cc. are indicated by words printed all in capital (Used for culture of coprophilic proletters.) tozoa. ) Peat agar. (From v. Wettstein, 1921; and Belar, 1928.) Solution A: CHEMICAL FORMULAS OF INORGANIC COMPOUNDS IN CULTURE MEDIA Ammonium phosphate 0.2 gram Magnesium sulfate, crystal. 0.05 gram Ammonium nitrate Calcium chloride NH.NOo 0 . 0 5 gram Ammonium phosphate Calcium sulfate (NñJ|)2 HPOl). 0 . 0 5 gram Calcium chloride Potassium phosphate, CaCl2 Calcium hydroxide dibasic 0 . 0 5 gram Ca(0H)2 Calcium nitrate, crystal. Water 1,000 cc. Ca (NOo) 2 . ^ 2 0 Calcium phosphate, monobasic Ferric chloride sol., 1$ 1 drop CaHj. (P0^)2 Calcium phosphate, tribasic Solution B: Ca3(P0jt)2 Calcium sulfate Peat 250 grams CaSOij. Ferric chloride Water 1,000 cc. FeClo Ferric sulfate Boil 2 to 3 hours, filter, dilute Fe 2 (S04) 3 Ferrous sulfate, crystal. with distilled water, if necessary, to FeS04.7H20 Hydrochloric acid a light coffee color. HCl Magnesium chloride, crystal. To make up media: MgCl 2 .6H 2 0 Magnesium phosphate, tribasic 100 cc. A + 100 cc. B + 1 to 4 grams Mg 3 (P0 4 ) 2 Magnesium sulfate agar. MgSOi^ Magnesium sulfate, crystal. Used for chrysomonads, pigmented cryptoMgSOi).. 7H 2 0 Potassium carbonate monads and euglenoids, certain small K2CO3 Potassium chloride freshwater dinoflagellates, etc. KCl

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA F o r marine forms: Some species, belonging to groups with freshwater forms cultivatable o n agar, can be grown on to 2$ agar with ALLEN SOLUTION. ALLEN SOLUTION, for marine forms. (From Belar, 1928.) Solution A : 5$ potassium nitrate in distilled water. Solution B : 1. Sodium phosphate, dibasic 4 grams 2. Calcium chloride 4 grams 3. Ferric chloride 2 grams 4. Hydrochloric acid 2 cc. 5. W a t e r 80 cc. Put water for B in three parts in three containers; dissolve salts separately. Add solution B , 1 drop b y drop to solution B , 2. Add acid and then add ferric chloride. F o r use: 1 liter seawater, 2 cc. solution A, 1 cc. solution B. AMOEBA MEDIUM. Small amoebae from soil and water m a y be g r o w n o n agar plates. (Alkaline Knop agar or bouillon or meat extract AGAR MEDIA have been recommended.) Various freshwater amoebae of moderate size m a y develop in established cultures of filamentous algae and diatoms. Amoebae of proteus type. These m a y be grown in finger bowls or moist chambers where the water depth is not too great. The amoeba m e d i u m of Chalkley (1930) is: Prepare a fluid m e d i u m as follows: Sodium chloride 0.1 gram Potassium chloride 0.004 gram Calcium chloride 0.006 gram Water, glass-distilled 1,000 cc. (it is convenient to make up stock in 100 times the concentration and to dilute this as needed.) Put 250 cc. into a finger bowl or dish about 4 inches in diameter (or a proportionate amount in a larger container). Add 1 g r a i n of unboiled polished rice to each 50 cc. of water. Seed w i t h amoebae and cover. Food organisms, such as Chilomonas, must also be present. A good culture should develop in a few weeks. W h e n the rice grains have b r o k e n up, more m a y be added to m a i n t a i n the culture, which should last at least for several months. It m a y be advantageous, in starting a culture, to place a thin layer of 0.75$ agar o n the bottom of the container, and to embed the rice grains in this. (Brandwein,

19350

Instead of rice, wheat grains and pieces of timothy h a y m a y be added. Two

4

wheat grains and 3 inches of h a y pieces m a y be added to a finger bowl half full of water. The rice, wheat, or h a y should first be sterilized by autoclaving dry, or freed of other protozoa b y placing d r y in test tubes w h i c h are put in boiling w a t e r for 15 minutes. A culture for amoebae m a y be prepared w i t h SYNTHETIC SPRING WATER, put in a finger bowl to a depth of about a n inch, with addition of 3 or 4 grains of wheat, 5 or 6 grains of rice, or 5 or 6 one-inch pieces of hay. Chilomonas should be introduced. Marine amoebae. Certain marine amoebae have b e e n cultivated in wheat infusion in sea water, or in SYNTHETIC SEA W A T E R w i t h wheat grains i n the amount of 6 in a Petri dish. They m a y also be grown on agar plates prepared as follows: 100 grams wheat in 1,000 cc. sea w a t e r or SYNTHETIC SEA W A T E R autoclaved 20 minutes. Filter and add 15 grams agar, restoring volume to 1,000 cc. Autoclave 30 to 60 minutes. Put agar m e d i u m into cotton-plugged tubes, or into Petri dishes. AM0EB0Z0A Ooze o n the bottom of ponds, marshes, ditches, or lakes, or floating o n the surface, m a y yield a variety of species. Bogs and swamps are important habitats. Shelled rhizopods (Thecamoebae) often occur in association w i t h mosses, especially sphagnum, w h i c h m a y have a rich fauna. Mosses wholly or partly submerged, o r on banks, or in moist terrestrial situations m a y contain them. Penard (1907, 1935), whose studies of rhizopods are monumental, described his methods of collecting as follows: F o r collecting from shallow ponds, streams, and marshes a test tube (12 x 2 cm.) is closed b y the thumb, lowered to the level of the mud, allowed to fill so as to draw in ooze on the m u d surface, then raised and emptied into a receptacle. The process is repeated, sampling from various places where bottom ooze occurs, until the receptacle is full. After allowing settling for 2 or 3 m i n u t e s , the upper fluid is poured off and the sediment returned to the test tube, w h i c h is then corked. It m a y be kept cool b y wrapping in damp cloth. A f t e r r e t u r n to the laboratory, the contents are passed through a sieve with l/2 mm. m e s h into a container w i t h clear water. To collect in deeper water, Penard used a rectangular metal receptacle (opening 14 cm. x 6 - 7 cm.) fastened to a line b y a metal loop, with a one-pound weight o n the line about a m e t e r i n front of the receptacle. This was dropped to the bottom, towed very slowly, then

MATERIALS AND METHODS IN THE STUDY OF PROTOZOA raised. O n return to the laboratory the contents were emptied into a container. Eventually the water clears and a b r o w n ooze appears o n the surface of the mud. The ooze consists largely of diatoms, and in it rhizopods m a y occur. Collections with ooze m a y be placed in small dishes so that the depth is not more than 2 or 3 inches and there is a thin layer of ooze o n the bottom. Living rhizopods m a y be found on the surface of this bottom deposit, and can be kept in the aquaria for a long time if suitable food is present. Putrefaction should be avoided. Some Amoebozoa can be cultivated in aquaria w i t h algae and chilomonads, some m a y be g r o w n in AMOEBA MEDIUM, others in hay or wheat infusion (see ARCELLA). Small gymnamoebae and some small thecamoebae can be maintained o n A G A R MEDIA. B e l a r (1921) cultivated species of Chlamydophrys o n alkaline K n o p agar. ARCELLA This shelled rhizopod m a y be grown i n HAY INFUSION or in pond water to which 2 grains of wheat and l/2 gram of h a y have been added for each 100 cc. of water. It will also do well in the fluid AMOEBA MEDIUM w i t h about double the amount of organic material. Introduce Chilomonas or another food organism. A low bowl or d i s h w i t h shallow water should be used.

5

lng are added: 0.01$ Potassium nitrate O.OOI56 Potassium phosphate, dibasic O.OOOOI56 Ferric chloride 236 SOIL EXTRACT This m e d i u m has been used successfully for some photosynthetic marine dinoflagellates, and can be used also for other marine flagellates. (Barker, 1935-) B E N E C K E SOLUTION Formula from v. Wettstein, 1921. Ammonium nitrate 0.2 gram Calcium chloride 0.1 gram Potassium phosphate, dibasic 0.1 gram Magnesium sulfate 0.1 gram Ferric chloride 1 drop of 1$ solution Distilled water 1000 cc. Formula from Belar, 1928. Stock solutions: ammonium nitrate 25É calcium chloride 256 magnesium sulfate, crystal. 256 potassium phosphate, dibasic F o r use: 0 . 0 5 s o l u t i o n , 1 9 6 cc. distilled water plus 1 cc. of each stock solution. B0D0NID FLAGELLATES

BACTERIA-EATING

PROTOZOA

B a c t e r i a constitute the food of a large number of phagotrophic protozoa, and the problem of cultivating them is one of growing the b a c teria and avoiding conditions unsuitable to the protozoa. Too heavy bacterial growth, resulting from the presence of too m u c h organic m a t t e r in the medium, is detrimental to most protozoa. Some of the ordinary methods for laboratory maintenance are referred to in this manual. (See HAY INFUSION, CEREAL MEDIUM, WHEAT INFUSION, FLOUR HAY MEDIUM, LACKEY WHEAT MEDIUM, LETTUCE MEDIUM, MEAT EXTRACT, HYPOTRICHA.) The number of types of infusion suitable for cultivation is large and varied. Their recommendation is largely on empirical grounds, but as a beginning it is useful to have some recipes. F o r better controlled cultures d i l a t e s m a y be washed, then maintained in a previously sterilized balanced solution (PETERS MEDIUM, PHYSIOLOGICAL BALANCED SOLUTION) and fed o n bacteria cultivated on nutrient agar. Serratia marcesans, Pseudomonas fluorescens, Ps. ovalis, Aerobacter aerogenes, and Bacillus subtilis have, among others, b e e n used successfully in feeding ciliates.

B A R K E R MEDIUM for marine

dinoflagellates.

To aged sea water, which has been kept in the dark preferably for some months, the follow-

Cultivated in infusions rich i n organic material; or o n A G A R MEDIA, w h i c h must be kept moist. C E R E A L MEDIUM. 10 grams of Mead's or similar granular cereal boiled in 1,000 cc. w a t e r f o r 10 minutes. F o r use dilute 1 to 10, or stronger or weaker as desired. To m a i n t a i n cultures, add a small amount of the d r y granules at intervals of several weeks. G o o d for Peranema, Oxyrrhis, various ciliates, and other protozoa. F o r marine and b r a c k ish w a t e r forms, the m e d i u m should be m a d e u p i n water of appropriate salinity. CHIL0M0NAS Chilomonas is v e r y common in f r e s h w a t e r ponds, especially where there is decaying v e g e tation. It can easily be obtained b y bringing in w a t e r and vegetation and letting it stand in the laboratory. If desirable, the w i l d culture m a y be enriched b y adding wheat grains or other nutriment. The flagellate m a y be cultivated readily i n m a n y media: EGG-YOLK MEDIUM, LACKEY W H E A T MEDIUM, soil-cheese m e d i u m , a m e d i u m of Pringsheim consisting of 1 gram peptone and 2 grams sodium acetate in a liter of double distilled

MATERIALS A N D METHODS IN THE STUDY OP PROTOZOA water, wheat infusion prepared b y adding 4 grams of wheat to 100 cc. of pond water, and various other infusions. Chilomonas is desirable as a food organism for amoebae, hypotrichs and some other ciliates. It can be maintained separately, and used for inoculation of m e d i a in which the predators are to be cultivated. CHLOROMONAD FLAGELLATES Cultivated in KNOP SOLUTION or SOIL E X TRACT. CHRYSOMONAD FLAGELLATES. M a y be cultivated on AGAR MEDIA Knop) or in S O I L EXTRACT.

(peat,

CILIATA Some free-living ciliates feed o n diatoms or other algae, and occur in relatively clear water, living in association with the plants. M o s t ciliates feed on bacteria, and occur in the largest populations w h e n bacteria are abundant. T h e y occur abundantly in association w i t h decaying organic matter. Bundles of grass or other plant materials, or sections of cane m a y be placed in bodies of water, and in the early stages of decay bacteria become abundant and populations of ciliates develop with them, and so can easily be secured. Ciliates occur in the soil and in mosses where the amount of moisture m a y be more or less restricted. They are in encysted stages w h e n environmental conditions are unsuitable for activity. Special faunas of ciliates occur in slime from decay on the bottom of deeper lakes, and in salt marshes associated w i t h purple bacteria and decaying algae. Attached ciliates, including stentorids and peritrichs, m a y be found on algae and on the stems and leaves of water plants. A large variety of sessile ciliates occur o n the bodies of aquatic animals, including fish, amphibia, and invertebrates of m a n y groups. Plankton tows of large bodies of fresh w a t e r usually will take fewer ciliates than algae and flagellates, but often some v e r y interesting sorts are obtained. The plankton of the sea contains a great variety of tintinnids, and a few are to be found in freshwater lakes. M a n y ciliates can readily be cultivated in the laboratory, using methods such as those referred to under BACTERIA-EATING PROTOZOA. CITRIC ACID Addition of 0.2$ to 1% to culture media has sometimes b e e n recommended for pigmented euglenoid flagellates and other forms where suppression of bacterial growth is desired.

6

C0LP0DID CILIATES Ciliates of the colpodid group (including Colpod^t and Tillina) m a y form cysts and excyst readily, so they are useful for study of this phase of development. They can be g r o w n in the fluid m e d i a for BACTERIA-EATING PROTOZOA. To control cyst formation, the ciliates m a y be kept in Syracuse watch glasses in hay infusion. W h e n after two days or so they are numerous, the water is evaporated slowly by exposure to air. After the culture is dry, new h a y infusion is added; then excystation m a y take place. The processes of multiplication, drying, and adding new medium m a y be repeated one or more times in the same dish. A concentrated culture of the ciliates m a y be allowed to dry in a dish the bottom of w h i c h is covered with a piece of filter paper. The cysts of Colpoda o n the filter paper can be kept dry Indefinitely, and will excyst w h e n put into h a y infusion. Processes of dedifferentiation and reorganization can readily be studied in these ciliates. COMMERCIAL FERTILIZER Various commercial fertilizers added to the water provide a convenient means of cultivating m a n y photosynthetic flagellates and algae. Rice (1946) found those with the formula 4-10-4 best for the largest number of forms; one gram of fertilizer m a y be used to one liter of fluid, or weaker concentrations m a y be desirable. The fertilizer is m i x e d in spring water; then the fluid heated to 80° C. and filtered. The filtrate is sterilized, inoculated after cooling, and kept covered in good light. Subcultures are prepared at intervals of several weeks, or a small amount of fertilizer m a y be added at intervals . Harris (1941) grew flagellates and algae in wooden casks sunk in the soil, filled with tap water, and enriched b y phosphate and potassium salts. Chlamydomonas, Euglena, and Pandorina flourished with addition at intervals of n i t r o g e n in the form, respectively, of ammonium n i trate, ammonium sulfate, and dried egg albumen. C0PR0PHILIC

PROTOZOA

A group of amoebae, flagellates, and ciliates that occur in water, soil, and sewage are capable of living and multiplying in fecal m a t ter. Some can multiply only after dilution, others develop in undiluted feces, fluid or n o r mal. T h e y either pass through the alimentary canal as cysts, or contaminate the feces after passage. Feces of certain animals constitute a more or less consistent source of certain forms. They are cultivated b y inoculating fecal samples into appropriate fluid m e d i a (especially those for BACTERIA-EATING PROTOZOA) or by streaking plates of AGAR MEDIA. The bionomics of coprophilic protozoa has b e e n reviewed by W a t s o n (1946).

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA DIDINIUM NASUTUM. This is a predacious ciliate which feeds chiefly o n Paramecium, and it can be maintained b y providing Paramecium caudatum for food. Care m u s t be exercised not to contaminate Paramecium cultures which are to be kept for that ciliate, as the population can be quickly destroyed by the predator. In one 24-hour period, the generations of Didinium resulting from a single individual at the beginning of the period m a y consume 60 or more Paramecium. Beers recommended growing Didinium in w a t c h glasses in 0.01$ KNOP SOLUTION, buffered by adding 5 cc. of Na0H-KH 2 P0j| buffer mixture of pH 6.8, to w h i c h a concentrated mass of Paramecium (obtained b y centrifuging) is added. W h e n food is exhausted Didinium encysts, and the cysts will remain viable in water for several years, but they will not withstand drying. The ciliate m a y be kept b y first feeding a culture in a vial, obtaining cysts in the vial, then stoppering and storing the vials halffilled with fluid. Excystation will take place w h e n h a y infusion from a Paramecium culture is added. DIMASTIGAMOEBA GRUBERI (NAEGLERIA GRUBERl) The organism was found b y Wilson (1916) in cultures prepared b y placing about 10 cc. of soil from the border of a creek in a dish with creek water or culture medium. It may be found in infusions prepared from moss or leaves or in macerations of hay. It was cultivated by W i l s o n in a liquid m e d i u m of creek water with 1% ground cabbage and cracker, equal parts, sterilized in an autoclave, and water added for increased fluidity. Rafalko (19^7) found that it grew best in a medium of pond water with strained human fecal material. It m a y be cultivated o n AGAR MEDIA as meat extract agar and Musgrave and Clegg agar medium. Pietschmann ( 1 9 2 9 ) used also peptone. F o r preparations showing the transformation to flagellates a piece of agar the size of a cover glass m a y be cut out, placed on a glass plate, and flooded with tap water. After transformation, fixed preparations m a y be made b y covering the agar with a cover glass and removing agar and cover to a dish of Schaudinn fluid. A f t e r 20 minutes the cover glass m a y be removed and placed for about 10 minutes longer in the fixative. Preparations for amoebae m a y be made from agar plates b y placing cover glasses on the surface and leaving for a period, then cutting out the agar around the glass and placing both together into the fixative, removing the cover glass after a period and post-fixing as before. Observations of the transformation of amoebae to flagellates m a y be made by placing amoebae into water in a hanging drop; after the desired stage is reached, the cover glass with the drop m a y be removed to a slide, exposing

7

first to osmic vapor if killing is desired. Rafalko (1947) kept the organisms in fluid cultures in Petri dishes, placing cover glasses o n the bottom of the dishes. W h e n a cover glass bearing amoebae was removed and flooded w i t h pond water, flagellates appeared in from 35 to 90 minutes or more. Adding 2 or more drops of a suspension of feces promptly induced transform a t i o n of the flagellates into amoebae. DINOFLAGELLATES F r e s h w a t e r dinoflagellates m a y be found i n lakes, ponds, and other bodies of water including pools and water troughs. T h e y m a y be t a k e n directly in water samples, or pressed out from among water plants. The larger plankton forms m a y be caught with a net of the finest silk bolting cloth; smaller ones can be concentrated b y centrifuging. Salt m a r s h pools and ditches m a y contain representatives of various genera, including Oxyrrhis, Amphidinium, and Exuviaella. Some species of Amphidinium, as well as other dinoflagellates, are sometimes found on wet sands that are uncovered at low tide. The dinoflagellates of marine plankton are abundant and varied; sometimes the water is discolored b y them, and luminescence m a y appear in the dark. They can be concentrated by a plankton net or b y centrifuging. Oxyrrhis can easily be cultivated in Infusions in sea water, or can be fed on diatoms. Some small freshwater dinoflagellates have' b e e n cultivated on peat-AGAR. F o r cultivation of certain marine species see BARKER MEDIUM. DUNALIELLA Dunaliella salina and D. viridls are small pigmented flagellates w h i c h occur in sea w a t e r of high salinity, and are often found in salt pans where sea water is being evaporated to m a k e salt. They m a y be kept in ALLEN SOLUTION in sea water, to which 10$ sodium chloride has b e e n added. F o r other culture methods, see F L A G E L LATES OF BRINE. Dunaliella m a y be u s e d as a food organism for marine animals. EGG Y O L K MEDIUM Prepare a paste by grinding 0 . 5 gram of hard-boiled egg yolk in a small amount of distilled water. Prepared dehydrated egg yolk m a y also be used. Add 500 cc. of distilled water. Let it stand two days and inoculate w i t h the protozoan desired. This is good for Chllomonas, subculturing every month. Paramecium and other ciliates do well. Chllomonas m a y be provided for ciliates using such food. EUGLENA Various m e d i a are suitable for cultures that are not bacteria-free:

MATERIALS AND METHODS IN THE STUDY OF PROTOZOA 1$ peptone in Vjh citric acid. Pea infusion made by "boiling ^0 split peas in a liter of water, with or without addition of some citric acid. One gram of malted milk in a liter of water. 6 grains of rice and 1 split pea boiled in 200 cc. water until the rice cooks to pieces. Pour off fluid and let stand for a day or more before inoculation. To 100 cc. KLEBS SOLUTION add 40 rice grains (boiled 5 to 10 minutes) and 900 cc. distilled water. Let stand 5 days. Put in good light and inoculate with Euglena. After 10 days, add 10 cc. KLEBS SOLUTION and 10 milligrams tryptophane powder to accelerate growth. Add 5 grains boiled rice each month. (Brandwein, 1935.) Euglena gracilis and some other species may be grown in ZUMSTEIN MEDIUM.

8

may be kept in cotton-plugged small flasks or test tubes, in which they have been sterilized dry or the tubes put in boiling water for fifteen minutes. Feeding may be accomplished by sprinkling flour on top of cultures, for many types of protozoa in an amount of about l/2 gram to 1,000 cc. Do not add too large an amount of food. If that is done the culture may be killed by too much bacterial growth. Some forms live in the presence of less organic matter, and it will be sufficient to add fresh materials when those already present have disappeared. As time goes on, the accumulation of waste products .may be detrimental. It may be advantageous at times to remove half the old culture fluid and replace it with fresh medium. FLAGELLATA (See also DINOFLAGELLATES)

Flagellates differ in their environmental requirements, from those living in situations where there is much decay to those requiring In addition to the methods given for Eugclear, well oxygenated water. They may be lena, pigmented forms may be cultured in SOIL EXTRACT, on peat AGAR, in 0.5$ ereptone solution looked for in any body of water, fresh, brackish or salt, from that of a hoof print or a spray+ 0.02$ potassium phosphate,vmonobasic + 0.01$ magnesium sulfate, or in LEFEVRE MEDIUM. Menoi- filled depression in a rock at the sea shore to a lake or the open sea. Pigmented flagellates dium and other heterotrophic forms will develop may become abundant enough to impart a green or in soil-cheese medium, or may be cultivated in other color to the water. Some flagellates, invarious organic infusions. Scytomonas can be cluding certain chrysomonads, choanoflagellates, cultured on alkaline Knop AGAR. Bacteria-free and bicocoecids, are attached to objects in the strains of euglenoids can be cultivated in the water or to the bodies of small plankton infollowing media: vertebrates . Fluid medium: Difco tryptone, 10 grams; sodium acetate, 0.25-0.5 grams; yeast extract, Larger flagellates, including dinoflagel0.25-0.5 grams, distilled water, 1000 cc. lates, and some colonial chrysomonads and phytomonads, may be taken in plankton tows with a net Agar slants: Difco starch agar, 20 grams; of the finest silk bolting cloth. For others, Difco tryptone, 5.0-7.5 grams; sodium acetate, samples of the water with ooze and material 0.5 grams; distilled water, 1000 cc. pressed out of submerged plant growth may be For a detailed discussion of culture of euglenoids, see Mainx, 1 9 2 8 and Pringsheim, 19^6. brought to the laboratory. Pigmented flagellates may aggregate on the lighted side of the container. Flagellates may be concentrated by centrifuging, or captured on floating cover FEEDING OF CULTURES glasses. Many non-pigmented flagellates will increase in numbers as decay takes place of orIn cultures there is an increase of the ganic matter, present in the collection or added. population followed by a decline. Maintenance Enrichment cultures may be prepared in various of the culture is primarily a question of food fluid media or, for some flagellates, on agar supply. To provide the best conditions for growth, transfers to fresh medium should be made plates. Flagellates that do not tolerate putrefaction may increase in population in flasks of at intervals. Many protozoa can, however, be SOIL EXTRACT, with or without a bottom layer kept in the same container for a long period of time if food is added at intervals. Flourishing of soil. Paramecium cultures have thus been kept for more Silicoflagellates are chiefly marine plankthan two years. ton forms. Skeletons are present in certain sedimentary rocks, usually along with diatoms. For this purpose small amounts of the same The fossils will be isolated by the customary substances used in making the infusion can be added at intervals of two weeks or a month or treatment of these rocks with boiling acids or so. Other substances may be equally suitable. other reagents, as mentioned under RADIOLARIA. Grains of wheat or rice, granular cereals, split peas, alfalfa, hay, dried and powdered lettuce, FLAGELLATES OF BRINE boiled lettuce leaves, crumbs of dried bread, sometimes pieces of meat or fish may be added. Flagellates which occur in water of salt For use in feedings, most of these substances lakes and marine pools in which the salt conEUGLENOID FLAGELLATES

MATERIALS AND METHODS IN THE STUDY OF PROTOZOA centration m a y be higher than sea water are cultivated in the following solutions (Ruinen, 1938): Water Sodium bicarbonate Potassium phosphate, dibasic Magnesium chloride Potassium nitrate

100 cc. 0.1 gram 0.02 gram 0.02 gram 0.02 gram

II Water Potassium phosphate, dibasic Magnesium chloride Sodium bicarbonate Sodium sulfide

100 cc. 0.05 gram 0.1 gram 0.5 gram 0.05 gram

Add to both m e d i a sodium chloride from J0> to saturation according to the salinity of the original habitat. A little SOIL EXTRACT m a y be added to the first medium. For Dunaliella viridis, Baas-Becking (1930) found it desirable to adjust the pH by means of potassium phosphate, dibasic, to about 9These m e d i a m a y also serve for culture of certain brine rhizopods and ciliates. FLOUR-HAY MEDIUM

9

Tests of Foraminifera m a y be washed u p b y the waves onto the beaches, and in appropriate localities m a y be found i n enormous numbers above the line of receding water. Tests m a y be collected from the bottom b y various types of apparatus suitable for bringing up portions of the sediment i n deeper water. Bottom-samplers capable of bringing u p u n d i s turbed cores a few inches in length make possible study of the position of tests in bottom deposits. (Phleger, 1946.) Fossil Foraminifera are present in m a n y marine sedimentary rocks. They are often abundant and have important practical uses as indicator fossils. Friable deposits, such as chalk, clay, and shales, m a y be disintegrated b y soaking in water. Shales m a y disintegrate after soaking for several days. If the rock does not break up, a little sodium hydroxide m a y be added and the material boiled. Fine sand and m u d in collections m a y be washed out through several successively f i n e r sieves (as 80 meshes to the inch, 40 meshes to the inch, and fine silk bolting cloth). "The coarser sieves retain the coarser material, and tests of larger Foraminifera m a y be retained. The strainings are dried in the air (if speed is necessary, w i t h the aid of heat), and the m a terial is spread out over a dark background and examined by strong reflected light.

0.1 gram chopped h a y and 0.13 gram white flour boiled ten minutes in 100 cc. spring water. HAHNERT SOLUTION W h e n 24 hours old, the infusion is diluted with an equal amount of additional spring water. The formula for a solution recommended b y Hahnert (1932) to cultivate amoebae of the proteus-group is: F0RAMINIFERA Living non-pelagic Foraminifera m a y be collected in the littoral zone by washing seaweeds, eelgrass, sponge growths, or other substances o n which they live. The material m a y be shaken and pulled apart in water in a bucket over a suitable sieve, through which the debris is strained. The washings w h i c h settle to the bottom contain Foraminifera, sand, and other fine materials. In taking it to the laboratory, it should be kept cool. To attempt cultivation, washings m a y be placed in 5 cc. amounts in finger bowls filled w i t h sea water, or in large moist chambers. The water m a y be changed every day (if necessary twice a day at first) until cultures become established, after which it should be changed occasionally. A growth of diatoms on the substratum is necessary. Diatoms m a y be grown separately in ALLEN SOLUTION. Small species of Nitzschia are good for food. Pelagic Foraminifera m a y be collected in surface marine tows b y plankton nets of 150 or more strands to the inch, and subsequently preserved in 70% alcohol or dried. Formalin should not be used for preservation unless neutral; acid formalin will dissolve the tests. (Phleger, 1940.)

Calcium chloride Potassium chloride Magnesium phosphate, tribasic Calcium phosphate, tribasic Calcium phosphate, monobasic Water

0.004 gram 0.004 gram 0.002 gram 0.002 gram 0.002 gram 1000 cc.

In cultivation of Pelomyxa, W i l b e r (1942) doubled the amount of each of the salts, except that 0.005 gram of magnesium phosphate was used. Another solution u s e d by Hahnert is the same as the first except for omission of tribasic calcium phosphate. Amoebae could also be cultivated in a solution with 0.08 gram sodium chloride and 0.004 gram sodium bicarbonate in addition to all the salts listed above, but they seemed to do b e t t e r i n the absence of the sodium salts. Kudo (1946) stated that for cultivation of Pelomyxa carolinensls Hahnert solution has no advantage over double-distilled water. H A Y INFUSION H a y yields nutrients and g r o w t h substances w h i c h are suitable for development of m a n y protozoa, so that h a y infusion is widely u s e d as a

10

MATERIALS AND METHODS IN T H E STUDY OF PROTOZOA basic culture medium. Timothy h a y Is regarded as the best type, but dried grasses of other kinds are also suitable. Rice stalks m a y be used. For a h a y infusion up to 6 grams of h a y to a liter of water can be used. More than this m a y reduce the pH below the lethal point or yield substances that are toxic. If other substances are added to the h a y (as wheat or flour) the amount of h a y must be reduced. A n infusion favorable for m a n y ciliates is prepared from 1 or 2 grams of h a y to a liter of water. As the nutrient properties of the m e d i u m decline, after a few weeks, food substances m a y be added. A h a y infusion m a y be made u p much more concentrated than this (as in amount of 10 grams to a liter), autoclaved and kept sterile, then diluted w i t h water before use. For observation of the succession of m i s cellaneous protozoan populations in a h a y infusion the following procedure m a y be followed: Boll 2 grams of h a y in water and add enough water (distilled or natural) to make 500 cc. Put in a dish or jar in the bottom of w h i c h is a layer of good soil. The h a y should be left in the fluid. K e e p covered. The culture m a y be seeded with a small amount of material from m i x e d laboratory or wild cultures, to start w i t h as much a variety of forms as possible. Examine the infusion at intervals b y taking a sample from the surface and another from the bottom. For a surface sample, a cover glass m a y be floated over night. Record as exactly as possible the types of organisms found each time, at top and bottom. Examinations m a y be continued for ten weeks or more, without adding anything to the culture. HELI0Z0A

stalks in 4 parts of pond water. The next day. a little dried yeast is added and the m e d i u m inoculated with Chilomonas. After 3 - 5 days it is inoculated with Stylonychia. Other culture m e d i a were WHEAT INFUSION and hay-wheat infusion. The last, which gave excellent results, was prepared b y adding wheat grains to the h a y before heating, and omitting yeast. Tryptone solution and rice infusion m a y be used for hypotrichs such as Oxytricha (Kay, 1945), bacteria serving as food. Cultures can be kept u p b y occasional addition of small amounts of nutriment. Turner (1930) recommended cultivating Euplotes patella in a m e d i u m made from 5 grams timothy hay, 10 halves of split peas, and 10 grains of wheat in a liter of water. After heating to the boiling point, the m e d i u m is set aside until the next day, then inoculated w i t h Chilomonas. A few days later the m e d i u m is ready for introduction of Euplotes. Hammond (1937) m o d i f i e d this m e d i u m b y boiling 5 grams timothy h a y and 3 grains of wheat in 50 cc.of Chalkley's solution (see AMOEBA MEDIUM), a n d adding when cool to 250 cc. of the solution. He also used wheat infusion prepared from 10 grains of wheat to the 300 cc. of C h a l k l e y 1 s solution. Chilomonas and Aspidisca served as food for Euplotes patella. K a y (19^-5) obtained cysts of Oxytricha b y concentrating the ciliates from a rich culture i n a small container, either in the original m e d i u m or in tap water. W i t h i n 24 hours m a n y of them had formed cysts. A cover could be sealed o n the container and the cysts stored. KLEBS SOLUTION.

M o d i f i e d solution B . (Brandwein, 1935.)

Potassium nitrate Magnesium sulfate Potassium phosphate, monobasic Calcium nitrate Bacto Tryptophane, powdered D i s t i l l e d water

0 • 25 gram 0 .25 gram 0 .25 g r a m 1 .00 gram 0 .10 gram 1 ,000 cc.

Actinophrys is often encountered in wild freshwater cultures, and it can be maintained in m e d i a for BACTERIA-EATING PROTOZOA with Chilodonella or some other small ciliate on whic.h it will feed. K N 0 P SOLUTION Actinosphaerium m a y be found in small permanent ponds and marshes, and will develop in Stock: Four separate solutions. (From pond water with 4 grains of wheat per 100 cc. Belaf, 1928.) It m a y be grown in a suitable fluid with grains 1. 10$ calcium nitrate, crystallized. of rice or wheat to w h i c h Paramecium or other 2. 5$ potassium nitrate. large ciliates, Stenostomum, or rotifers are 3. 5$ magnesium sulfate, crystal. added as needed. 5$ potassium phosphate, monobasic. Acanthocystids have siliceous skeletal eleDilution for u s e : ments, and should do well i n ARTIFICIAL SPRING 1$ solution: 150 cc. distilled water; WATER. 10 cc. of calcium nitrate; 5 cc. of each of others. The last is added drop by drop while shaking. A drop of 0.1$ HYP0TRICHA ferric chloride solution m a y be added if needed. Hypotrichs such as Stylonychia often appear 0.05$ solution: 10 cc. of 1$ solution, late in the cycle of uncontrolled h a y infusion 190 cc. of distilled water. cultures. Chen (19^4) cultivated Stylonychia in 0.01$ solution: 40 cc. of 0.05$ soluh a y infusion made b y boiling 1 part of rice tion, 160 cc. of distilled water.

11

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA LACKEY WHEAT MEDIUM 25 cc. water and 5 grams wheat put in large test tute. Tube plugged, autoclaved 2 hours. • Various percentages of this stock medium are used for different flagellates, ciliates, and amoebae. Excellent for Entosiphon and Peranema. (Lackey, 1 9 2 7 . ) LEFEVRE MEDIUM

(From Hollande, 1942.)

Distilled water Potassium nitrate Potassium phosphate, dibasic Magnesium sulfate Calcium nitrate Ferric chloride solution For certain flagellates add: Peptone For euglenolds add SOIL EXTRACT or sphagnum extract

1,000 cc. 0.2 gram 0.04 gram

0.02-0.03 gram 0.1-0.4 gram 1 drop 0.125 gr. 15-20 cc.

LETTUCE INFUSION Lettuce leaves are dried in an oven until they are crisp or brown, but they must not be burned. The dried leaves are powdered and the powder may be stored. Lettuce infusion is prepared by boiling 1.5 grams of lettuce powder in a liter of distilled water for five minutes. The infusion may then be filtered into small flasks and autoclaved. It can be kept while sterile for long periods. For use, one part distilled water Is added to two parts lettuce Infusion. Wichterman (194-9) reported that all species of Paramecium do well in lettuce infusion, though for all freshwater species except P. bursaria the cultures do not last so long as in hay infusion. For P. calkinsi and P. woodruff! from brackish water, sea water Is used in the infusion to the amount of the salinity of the original habitat. Hyman (1941) recommended using whole lettuce leaves which have been prepared by bringing to a boil. A lettuce leaf to a jar is satisfactory for Paramecium and other protozoa. Amoebae and other rhizopods may be cultivated in an inch or two of water with a piece of lettuce leaf. LIGHT Autotrophic flagellates must be sufficiently Illuminated, but must be protected, when in small vessels, from too much direct sunlight, which will raise the temperature excessively. The best light is that of a north window. A controlled illumination can be provided by means of an electric light source, with circulating water to carry off the heat. (See Belar, 1928, and Pringsheim, 1946.)

Heterotrophic protozoa do as well, if not better, in the dark as in the light, and can best be kept at least in much subdued light. Subdued light or darkness may be an advantage for impure cultures in keeping down the growth of algae. MEAT EXTRACT 0.025$ solution of prepared beef extract is a good medium for cultivation of bacteria-eating protozoa and also for other flagellates and algae. It may be neutralized to litmus by means of sodium or potassium carbonate. For euglenolds, 0.001$ to 1$ beef extract with 1$ citric acid has been used. Bouillon: 125 grams of lean meat are cut up and cooked an hour in 1,000 cc. of distilled water. The scum is removed, the fluid filtered, and 1 gram of peptone added to 100 cc. of bouillon. The mixture may be cleared and neutralized if necessary with a 5$ solution of egg-white. For use: 10 cc. bouillon and 90 cc. water. For Bouillon agar add 1$ to 2$ agar. M0LISCH SOLUTION Ammonium phosphate Potassium phosphate, dibasic Magnesium sulfate, crystal. Calcium sulfate Ferrous sulfate, 1 drop 1$ solution to

0.08$ 0.04$ 0.04$ 0.04$ 100 cc.

MOORE SOLUTION Ammonium nitrate Potassium phosphate Magnesium sulfate Calcium chloride Ferric sulphate Water

0 . 5 gram 0.2 gram 0.2 gram 0.1 gram trace 1000 cc.

For blue-green algae, the amount of ammonium nitrate should be doubled, and 1$ to 2$ of glucose may be added with benefit. An agar medium may be prepared by adding 5 grams of agar to a liter of Moore solution heated to boiling, and pouring into plates or 100 cc. Erlenmeyer flasks. MYCET0Z0A Slime molds may be found in the proper season on moist decaying logs and bark, on compost, about manure, under wet leaves, and in other situations where there is decaying vegetable matter and moisture. Some Mycetozoa which feed on bacteria have been cultivated on alkaline Knop AGAR and beef extract agar plates. Others can be kept on a substrate of plain 2$ agar and fed by ground rolled oats or by streaking a suspension of yeast.

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA Oatmeal agar can be prepared as follows: A medium Is made up of 2$ agar in distilled water, buffered at pH 6. Either place 0.5 gram of ground rolled oats on the bottom of a Petri dish and pour 20 cc. of the agar over it; or cook 30 grams of ground rolled oats in the liter of agar, autoclave, and use in pouring plates. Large Plasmodia can be kept on wet paper and fed by adding ground rolled oats or rice. A Petri dish wrapped in filter paper and placed in shallow water in a moist chamber provides a good substrate. The ground food is given daily, in amounts not more than equal to the bulk of the Plasmodium, by sprinkling a small amount around and on top of the organism. Cultures are best kept in the dark in a cool place. Temperatures of 20° to 220 c. are optimum for most species. When the substratum dries the amoeboid forms pass into the resistant forms of sclerotia. In old cultures where food is deficient sporeforming bodies may occur. The spores germinate when placed on agar plates. (See Camp, 1936; Howard, 1931•) PARAMECIUM. (See also Wichterman, 1949.) Species of Paramecium are often found in infusions prepared from plant material from ponds and lakes. The ciliates may appear after a few days when the plants are put in a jar with enough water to cover them. The organic matter decays, bacteria grow rapidly, and as they increase various protozoa multiply. Addition of bread crumbs or other substances mentioned under FEEDING OP CULTURES will keep up growth. Paramecium may be isolated from other protozoa on depression slides, and as a population develops moved to larger containers. Certain species of Paramecium occur in brackish water. Various media for BACTERIA-EATING PROTOZOA are suitable for Paramecium. HAY INFUSION and LETTUCE INFUSION are good culture media. Cultures may be maintained in hay infusion to which wheat grains are added at intervals to keep up the food supply. The grains of wheat may be sterilized dry in test tubes in an oven or autoclave or by keeping the tube in boiling water for 15 minutes. Generally 20 to 30 grains to a liter of hay infusion will be adequate. Mas3 cultures for securing a large number of Paramecium may be made in battery jars or aquaria. The container is filled with hay infusion prepared by boiling 2 to 4 grams of hay in a liter of water. The hay may be placed in the medium, and some fresh hay, wheat, or other nutrient added as the population declines. A massing of the ciliates at the margins near the top may take place if the culture is stirred vigorously and the scum wiped down from the sides, and it is allowed to 3tand for 15 minutes or more. (Hyman, 1931.) Biological supply houses may be able to furnish single-species cultures of several species of Paramecium.

12

Conjugation and nuclear reorganization in Paramecium Conjugation in Paramecium caudatum was found in abundance by Calkins and Cull (1907) in material obtained by seeding a hay infusion with the ciliate, leaving it some weeks until there was a white band of Paramecium below the surface, then removing the ciliates from this white band to a fresh hay infusion in a watch glass. Conjugating pairs occurred in 1 or 2 days. Depletion of food after a period of plenty is an important factor Influencing the onset of conjugation in mass cultures. Crowding a flourishing culture may result in an epidemic of conjugation. (Giese, 1939.) The temperature influences conjugation, along with other factors. Giese found that 20°-26° C. was most favorable to conjugation in P. multimicronucleatum. Sonneborn (1936) found that mass cultures in fresh medium kept In small dishes at 31° C. were favorable to the occurrence of conjugation in certain stocks of Paramecium aurelia. Instead of conjugation, in these small dishes kept at 31° C., nuclear reorganization without conjugation (endomixis or autogamy) frequently was induced in Sonneborn's experiments. More significant in conjugation than the environmental factors are the internal factors of differentiation of mating types. There are varieties within species of Paramecium between which conjugation will not occur. Within a variety, there are two or more mating types, and conjugation will occur between any two different ones when they are brought together. The mating reaction may be kept under control for laboratory purposes in Paramecium bursaria. Different mating types may be kept in mass cultures in hay infusion or lettuce infusion. When the ciliates of two or more different types are mixed, agglutination will occur, forming groups of several or many ciliates, provided the animals are mature and in proper condition. (Jennings, 19390 This clumping may begin at once upon mixture; but in other cases of mixture it does not occur until later. After half an hour or more clumps begin to break up, and conjugating pairs appear, remaining united for 24 to 48 hours. PEAT EXTRACT 250 grams of peat boiled 2 or 3 hours in a liter of spring water or double-distilled water, filtered, and the filtrate diluted as desired. Sphagnum extract may be prepared from dried sphagnum in a similar way. PELOMYXA Pelomyxa carolinensis was cultivated by Kudo (1946) in laboratory finger bowls with 200 cc. of double-distilled water (Pyrex water), 2 rice grains, and Paramecium added as food. The organism may be kept in Chalkley solution or

MATERIALS A N D METHODS IN THE STUDY OP PROTOZOA Hahnert solution with rice grains and Euglena, Chilomonas, or other forms for food, in a similar m a n n e r to that in which amoebae of the proteus-type are cultivated. Kudo noted that u n favorable culture conditions exist when the temperature is above 22° C., when decomposing organic m a t t e r accumulates, w h e n the pH falls below 6.4 or rises above 8.2, or when predatory suctoria are present.

PETERS MEDIUM

(Hetherington, 1934a)

13

EXTRACT. F o r marine forms agar plates m a y be m a d e with ALLEN SOLUTION. F l u i d m e d i a that m a y be u s e d for various phytomonads are 0.05 - 0 . 1 $ Benecke solution, K n o p solution, and soil extract, or some c o m mercial fertilizer m a y be employed. Marine forms m a y be g r o w n i n A l l e n solution in sea water. Chlamydomonas was cultivated b y K a t e r (1929) in a weak alfalfa infusion. Illumination m a y be b y daylight or a n artificial light source. Morse (1943) cultivated Pandorina m o r u m i n 0.01$ Arlco peptone solution made u p w i t h f i l tered and boiled pond water.

Stock solutions: 1. Calcium bicarbonate. Shake 10 grams of calcium hydroxide, in a liter of Heterotrophic species. double-distilled water. Let stand, in a corked flask, 2 days, shaking at Polytoma and Polytomella m a y be cultivated intervals. Pass carbon dioxide in infusions of earth and manure, o n B o u i l l o n through the solution (at 23°C.) for agar, i n pure culture or on agar w i t h a special one hour. Cork and leave 24 hours. Polytoma-medium as follows: Sodium acetate Pour off the supernatant fluid into 0.2 gram; glycine, 0.2 gram; glucose 0.2 gram; glass-stoppered bottles, and pass potassium carbonate, 0.5 gram; magnesium sulcarbon dioxide through each f o r one fate, crystal, 0.01 gram; potassium phosphate, minute. dibasic, 0.02 gram; water 100 cc.; agar 0.75 to (See also H a l l , 1937-) 2. 0.015 M solution of magnesium sulfate. 1 gram. 3- 0.015 M solution of potassium phosphate, dibasic. PR0TE0MYXID RHIZ0P0DS 4. 0.0075 M solution of sodium phosphate, dibasic. These protozoa m a y be found in pools, i n To prepare a liter of medium mix 55 cc. of infusions, and o n agar cultures of manure, g a r solution 1 , 10 cc. of each of the others, and d e n earth, etc. Some forms live in association double-distilled water t o one liter. w i t h g r e e n or blue-green algae, as Oscillatoria. A simpler modification, usable for complex nutrient m e d i a , is given b y Hetherington (1934b) M a n y forms do well o n alkaline K n o p A G A R . Others can be cultured o n K n o p agar or peat-agar as : plates w i t h Chlorogonium or another suitable o r Calcium chloride 0.00055 M ganism to serve as food. T h e y m a y also be g r o w n Magnesium sulfate 0.00015 M in 0.01$ K N O P or B E N E C K E SOLUTION w i t h a suitPotassium carbonate 0.00015 M able organism as food. F o r f.orms living w i t h Sodium carbonate 0.000075 M filamentous algae, the algae can be m a i n t a i n e d i n a suitable medium. PHYSIOLOGICAL BALANCED SOLUTION (Osterhout, from Taylor and Strickland, 1935.) RADI0LARIA Water 1000 cc. Sodium chloride 10.4 grams Radiolarla are pelagic marine protozoa, and Magnesium chloride, crystal. 0.85 gram can be taken b y plankton nets i n the open sea. Magnesium sulfate, crystal. 0.4 gram Some forms occur only i n deep water. T h e y are Potassium chloride .23 gram delicate as living animals, and require care in Calcium chloride .1 gram bringing them in f o r study and in preservation. F o r use, dilute 100 times. T h e y cannot be cultivated or m a i n t a i n e d in the To each 300 cc. add 10 cc. M/20 sodium laboratory while living. M o s t Radiolarla have phosphate, dibasic, for buffering. Adjust pH skeletons. The siliceous skeletons form dew i t h M/20 sodium hydroxide. It will be satisposits on the bottom of the sea. Radiolarian factory for m a n y forms when a sample to w h i c h a ooze, consisting largely of these skeletons, drop of 1$ aqueous neutral red is added becomes forms a n extensive belt o n the sea bottom of the orange. equatorial Pacific Ocean. Fossil R a d i o l a r l a are important constituents of terrestrial sediments and rocks, though they are less abundant than Foraminifera. T h e y have had a part in the PHYT0M0NAD FLAGELLATES (See also DUNALIELLA, origin of ancient siliceous schists. Skeletons VOLVOX.) m a y occur in more friable rocks, including m u d Autotrophic species. stones and shales. Solitary freshwater forms m a y be grown o n AGAR plates (0.7$ agar) made with 0.05$ Isolation of fossil skeletons for study .BENECKE o r K N O P SOLUTION, or with PEAT or SOIL varies with the nature of the deposit. Friable

14

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA rocks m a y break u p w h e n soaked In cold or b o i l ing water. Deflandre (1947) recommended breaking material into small fragments and placing them in a hot solution of sodium hyposulfite, sodium sulfate, or sodium acetate. After some hours of impregnation, the preparation is a l lowed to cool. Crystallization m a y bring about disintegration. The process can be repeated as m a n y times as necessary, and the salt then washed out. Subsequent treatment with acids will remove organic material and non-siliceous fossils. Material from w h i c h skeletons are to be isolated m a y be covered successively with concentrated hydrochloric acid, nitric acid, and sulfuric acid and boiled gently for about 20 minutes i n each fluid. Between treatments the excess acid m a y be poured off o n the material w a s h e d in water. Treatment w i t h potassium chlorate or sodium hydroxide m a y also be given, to complete elimination of the organic matter.

S O I L EXTRACT Garden earth is boiled for a n hour w i t h the same volume of spring water and allowed to stand one or two days. The fluid is decanted and put into flasks, and kept sterile. F o r use it is diluted from 4 to 6 times with distilled water. M a n y pigmented flagellates, aa well as others if organic nutrients are present, m a y be g r o w n in the fluid over a layer of soil. A little lime m a y be added to give an alkaline reaction. (See Pringsheim, 1946.) SOIL PROTOZOA Most of these are bacteria-feeding forms and m a n y can be g r o w n o n AGAR MEDIA or in fluid m e d i a that are suitable for such forms. See BACTERIA-EATING PROTOZOA. A small amount of the soil collection (which m a y have b e e n kept dry for a long time) is put o n the plate or in the medium. A n agar plate m a y be covered with a layer of sterile water. SPIR0ST0MUM 1$ of Timothy h a y and 1 of wheat grains are boiled in spring water. A f t e r the solution is cool, one tablespoonful of fresh cow manure is added to a liter. I n 2 or 3 days, Spirostom u m from an old culture is added. Addition of cow manure to declining cultures brings up Spirostomum again. (Specht, 1 9 3 5 0 STENTOR A paste made b y grinding l/2 gram of hardboiled egg yolk in a little water is added to 750 cc. of distilled water. The mixture is allowed to stand for 3 days, filtered, and inoculated with Chilomonas and then with Stentor.

Stentor can be maintained in fluid cultures of various sorts for BACTERIA-EATING PROTOZOA, w h e n it feeds on bacteria; or it m a y be kept in a solution such as 0.01$ K N 0 P or 0.01$ B E N E C K E and fed o n Gonium or other suitable organism. See also WHEAT INFUSION. SUCT0RIA Most suctoria are sessile, and occur prim a r i l y on the surface of aquatic plants and animals. Filamentous algae, i n particular, m a y bear suctoria that can readily be found and studied. T h e y attach themselves to aquatic insects and their larvae, to copepods, to the stalks of peritrichs, to snail shells, and occur o n the gills of crustacea and fish. Certain suctoria (as well as spirochonids) occur on the gills of gammarid crustacea. Plants with attached suctoria m a y be placed in a container in the laboratory, where f r e e swimming stages develop and the young developing forms that attach themselves to floated cover glasses or immersed slides can be studied. Rieder (1936) obtained suctoria by immersing glass plates, about 3 inches wide by 7 l/2 Inches long, to w h i c h a row of 5 .microscope slides were attached by rubber bands, in w a t e r of the natural habitat. Upon removing the plate from the water, the slides were removed from under the rubber bands, and placed in a flask of water from the habitat, the several slides being separated b y replacement of the bands on them. I n this way, the slide could be transported to the laboratory w i t h the suctoria living, if too long transport w o u l d not be necessary. Otherwise the material could be fixed at the site of collection.

SYNTHETIC SEA WATER Distilled w a t e r Calcium chloride Magnesium chloride crystal. Magnesium sulfate crystal. Potassium chloride Sodium chloride Sodium bromide Sodium bicarbonate

1 ,000 1 .220 5 .105 7 .035 0 • 763 28 .340 0 .082 0 .210

cc. grams grams grams grams grams grams grams

SYNTHETIC SPRING W A T E R (1) Sodium metasilicate 15 Sodium chloride 12 Sodium sulfate 6 Calcium chloride 6. Magnesium chloride 3Ferric chloride 4 Dist. w a t e r 1000

mg. mg. mg. mg. mg. mg. cc.

(2) 100 mg 12 mg 6 mg 6.5 mg 3 - 5 mg mg. 4 1000 cc.

Adjust by hydrochloric acid to pH 6.8 to 7.0. The first formula is used in early transfers from nature; after that the second.

15

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA TETRAHYMENA GELEII, bacteria-free. H e t h e r i n g t o n 1 s medium: Difco yeast extract 0.5$ Difco powdered yeast 0.2$ Made up in PETERS MEDIUM Laboratory stocks of this ciliate in pure culture can be kept readily in 2$ proteosepeptone in cotton-plugged test tubes, w i t h 0.5$ dextrose added if desired, but this is not necessary for routine maintenance. Another suitable medium is: Yeast extract 0.5$ Dextrose 0.5$ V0LV0X Volvox minor and Volvox globator were maintained by Uspenski and Uspenskaja (1925) for some months with regular multiplication in a n inorganic m e d i u m with addition of iron, that addition being very important. The m e d i u m was made up as follows: Potassium nitrate 25 milligrams Magnesium sulfate 25 mg. Calcium nitrate 100 mg. Potassium phosphate, monobasic 25 mg. Potassium carbonate 34.5 mg. Ferric sulfate 1.25 mg. Water, double-distilled 1000 cc. The ferric sulfate should be sterilized separately and added after sterilization of the medium. Every ten days in summer 0.5 mg. of the iron compound was added; in winter the addition of iron was made once a month. The pH of the medium was 7.6. Cultures should be placed where they will receive a good light, as at a south window. Species of Volvox have been maintained in SOIL EXTRACT.

m e d i u m excysted in about half a n hour to a n hour, and conjugation phenomena occurred in l'ito 36 hours or more. W H E A T INFUSION Boil wheat grains in a small amount of water for 2 or 3 minutes. A d d the boiled wheat grains to the culture water in the number desired. Let it stand a day or more before inoculating w i t h protozoa. The number of grains that are used in a culture varies with the type of ciliate to be maintained. The following numbers are recomm e n d e d for different ciliates (Hyman, 1931) : Paramecium 60-70 to a liter Stentor 20 to a liter Vorticella 20 to a liter Hypotrichs 40 to a liter Wichterman (194-9) recommended boiling 50 grains of wheat in a liter of w a t e r for five minutes to cultivate Paramecium. F o r a weaker infusion the boiled grains are removed to f r e s h water. YEAST MEDIUM for excystation. Dissolve 1 gram of yeast extract paste in a Autoliter of PHYSIOLOGICAL BALANCED SOLUTION. clave if desired. (Taylor and Strickland, 1935.) ZUMSTEIN MEDIUM I Peptone Glucose Citric acid Magnesium sulfate, crystal. Potassium phosphate, monobasic Water

V0RTICELLA EGG-YOLK MEDIUM, WHEAT INFUSION, and other m e d i a for BACTERIA-EATING PROTOZOA m a y be used. Alfalfa-wheat culture (Finley, 1936): 2 grams alfalfa hay and 3 grams wheat grains boiled in 100 cc. distilled water 5 minutes. Filter, restore to 100 cc., autoclave. After cooling, add to 5 cc. of cooled nutrient 10 cc. of sterile spring water. Inoculate with A c h r o mobacter liquefasciens. Cysts placed in this

gram gram gram gram gram cc.

II Peptone 1.0 gram Glucose 0.4 gram Citric acid 0.4 gram Magnesium sulfate, crystal. 0.02 gram Potassium phosphate, monobasic 0.05 gram Ammonium nitrate 0.05 gram Water 100 cc. One or the other m e d i u m has b e e n used for certain species of Euglena.

REFERENCES ON COLLECTION AND CULTIVATION OF FREE-LIVING Baas-Becking, L.G.M., 1930. Observations o n Dunaliella viridis Teodoresco. Contributions to Marine Biology, Stanford: 102-114. Barker, H.A., 1935The culture and physiology of the marine dinoflagellates. Arch. Mikrobiol., 6:157-181.

0.5 0.5 0.2 0.02 0.05 100

PROTOZOA

Barker, H.A. and C.V. Taylor, 1933Studies o n the excystment of Colpoda cucullus. Physiol. Zool., 6:127-136. The culture of Didinium Beers, C.D., 1937nasutum in Galtsoff et al: Culture methods for invertebrate animals, pp. 100-103.

MATERIALS AND METHODS IN THE STUDY OF PROTOZOA Belar, K. , 1921. Untersuchungen über Theoamöben der Chlamydophry3-Gruppe. Arch. Protistenk., 43:287-354. Belar, K., 1928. Untersuchung der Protozoen in_ Peterfi: Methodik der wissenschaftlichen Biologie, vol. 1:735-826. Brandwein, P.P., 1935- The culturing of freshwater protozoa and other small invertebrates. Amer. Nat., 69:628-632. Calkins, G.N. and S.W. Cull, 1907. The conjugation of Paramaeciumaurelia (caudatum). Arch. Protistenk., 10:375-415. Camp, W.G., 1936. A method of cultivating myxomycete Plasmodia. Bull. Torrey bot. Club, 63:205-210. Chalkley, H.W., 1930. Stock cultures of ameba. Science, 71:442. Chen, Yueh-Tseng, 1944. Studies on the neuromotor systems of Stylonychia pustulata and Stylonychia mytilus. J. Morph., 75:335-345• Claff, C.L., 1940. A migration-dilution apparatus for the sterilization of protozoa. Physiol. Zool., 13:334-341. Dawson, J.A., 1928. The culture of large freeliving amoebae. Amer. Nat., 62:453-466. Deflandre, G., 1947- Microscopie pratique. Paris: Paul Lechevalier. Fauré-Fremiet, E., 1931- Quelques résultats obtenus avec la méthode des lames immergées. Bull. Soc. zool. Pr., 56:479-482. Finley, H.E., 1936. A method for inducing conjugation within Vorticella cultures. Trans. Amer. micr. Soc., 55:323-326. Galtsoff, P.S. et al. (ed.), 1937- Culture methods for invertebrate animals. Ithaca: Comstock Publishing Company. Giese, A.C., 1939- Studies on conjugation in Paramecium multimicronucleatum. Amer. Nat., 73:432-444. Hahnert, W.F., 1932. Studies on the chemical needs of Amoeba proteus : a culture method. Biol. Bull., 62:205-211. Hall, R.P., 1937- Growth of free-living protozoa in pure cultures in Galtsoff et al.: Culture methods for invertebrate animals, PP- 51-59Hammond, D.M., 1937- The neuromotor system of Euplotes patella during binary fission and conjugation. Quart. J. micr. Sei., 79: 507-557. Harris, T.M., 1941. Notes on the culture of freshwater algae. New Phytol., 40:157-158. Hegner, R., 1929- Methods for cultivating and fixing clones of Arcellas. Trans. Amer, micr. Soc., 48:214-215. Hetherington, A., 1932. The constant culture of Stentor coeruleus. Arch. Protistenk., 76: 118-129. Hetherington, A., 1933- The culture of some holotrichous ciliates. Arch. Protistenk., 80:255-280. Hetherington, A., 1933. The pure culture of Paramecium. Science, 79:4l3-4l4. Hetherington, A., 1934a. The sterilization of protozoa. Biol. Bull., 67:315-321. Hetherington, A., 1934b. The role of bacteria

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in the growth of Colpidium colpoda. Physiol. Zool., 7:618-641. Hollande, A., 1942. Etude cytologique et biologique de quelques Flagelles libres. Arch. Zool. exp. gen., 83:1-268. Hopkins, D.L. and P.L. Johnson, 1929- The culture of Amoeba proteus in a known salt solution. Biol. Bull., 56:68-73Howard, F.L., 1931. Laboratory cultivation of myxomycete Plasmodia. Amer. J. Bot., 18: 624-628. Hyman, L. H., 1925- Methods of securing and cultivating protozoa I. "General statements and methods. Trans. Amer. micr. Soc., 44: 216-221. Hyman, L.H., 1931. Methods of securing and cultivating protozoa II. Paramecium and other ciliates. Trans. Amer. micr. Soc., 50: 50-57. Hyman, L.H., 1941. Lettuce as a medium for the continuous culture of a variety of small laboratory animals. Trans. Amer. micr. Soc., 60:365-370. Ibara, Y., 1926. Culture medium for the ciliate Lacrymaria. Science, 63:212. Jane, F.W., 1942. Methods for the collection and examination of fresh-water algae, with special reference to flagellates. J. Quekett micr. CI., (4), 1:217-229Jennings, 1939. Genetics of Paramecium bursaria. I. Mating types and groups, their interrelations and distribution; mating behavior and self sterility. Genetics, 24:202-233. Kahl, A., 1930. Wimpertiere oder Ciliata (infusoria)! in Dahl: Die Tierwelt Deutschlands, 18. Teil. Jena: Fisher. Kater, J. McA., 1929- Morphology and division of Chlamydomonas with reference to the phylogeny of the flagellate neuromotor system. Univ. Calif. Publ. Zool., 33: 125-168. Kay, M.W., 1945- Studies on Oxytricha bifaria Stokes II. Cystic reorganization. Trans. Amer. micr. Soc., 64:267-282. Kidder, G.W., 1941. The technique and significance of control in protozoan culture in Calkins and Summers: Protozoa in biological research. New York: Columbia University Press. Pp. 448-474. Kudo, R.R., 1946. Pelomyxa carolinensis Wilson. I. General observation on the Illinois stock. J. Morph., 78:317-351Lackey, J.B., 1927- A culture medium for freeliving flagellates. Science, 6 5 : 2 6 1 . Lackey, J.B., 1936. Occurrence and distribution of the marine protozoan species in the Woods Hole area. Biol. Bull., 70:264-278. Mainx, F., 1928. Beiträge zur Morphologie und Physiologie der Eugleninen. I. Morphologische Beobachtungen, Methoden und Erfolge der Reinkultur. Arch. Protistenk., 60: 305-354. Mainx, F., 1929 in_Tabulae Biologicae, 5:1-23. (Summary with references to methods of culture of flagellates and algae.) Morse, D.C., 1943- Some details of asexual re-

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA production in Pandorina morum. Trans. Amer. raicr. Soc., 62:24-26. Mottram, J.C., 1943. A simple technique for cultivating ciliates. J. Quekett micr. CI., (4), 1:284-286. Penard, E., 1907- On the collection and preservation of fresh-water rhizopods. J. Quekett micr. CI., (2), 10:107-116. Penard, E., 1935- Rhizopodes d'eau douce. Recoltes, p r e p a r a t i o n s ^ . et souvenirs. Bull. Soc. francaise Microscopie, 4:57-73Phelps, A., 1934. Studies on the nutrition of Paramecium. Arch. Protistenk., 82:134-163. Phleger, F.B., Jr., 1940. Collecting pelagic Foraminifera. Science, 92:553-554. Phleger, P.B., Jr., 1946. Methods of study of recent Poraminifera. Report of the Committee on Marine Ecology as related to Paleontology, no. 6:79-80. Wash.: National Research Council. Pietschmann, K., 1929- Untersuchungen an Vahlkampfia tachypodia Gläser. Arch. Protistenk., 65:379-425. Pringsheim, E.G., 1915- Die Kultur von Paramaecium bursaria. Biol. Zbl., 35:375-379Pringsheim, E.G., 1936. Zur Kenntnis saprotroper Algen u n Flagellaten. I. Ueber Anhaüfungskulturen polysaprober Flagellaten. Arch. Protistenk., 87:43-96. Pringsheim, E.G., 1946. Pure cultures of algae: their preparation and maintenance. New York: Macmillan. Rafalko, J.S., 1947. Cytological observations on the amoeboflagellate, Naegleria gruberi (Protozoa). J. Morph. 8l:l-44. Rice, N.E., 1946. Commercial fertilizer in the culture of fresh-water algae. Science, 104:16-17. Rieder, J., 1936. Biologische und ökologische Untersuchungen an Süsswasser-Suktorien. Arch. Naturgesch., neue Folge, 5:137-214. Ruinen, J., 1938. Notizen über Salzflagellaten. II. Über die Verbreitung der Salzflagellaten. Arch. Protistenk., 90:210-258. Seaman, G.R., 1947Penicillin as an agent for sterilization of protozoan cultures. Science, 106:327.

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Sonneborn, T.M., 1936. Factors determining conjugation in Paramecium aurelia. I. The cyclical factor: the recency of nuclear reorganization. Genetics, 21:503-51^• Specht, H., 1935. The culture of Spirostomum ambiguum. Arch. Protistenk., 85:150-152. Stanley, J., 1946. A method of growing dense cultures of Paramecium. Science, 103: 115-116. Some Taylor, C.V. and A.G.R. Strickland, 1935factors in the excystment of dried cysts of Colpoda cucullus. Arch. Protistenk., 86:

181-190.

Trager, W . , 1937. Some methods for the pure culture of protozoa Iii Galtsoff et _al_. : Culture methods for invertebrate animals, pp. 112-119. Turner, J.P., 1930. Division and conjugation in Euplotes patella with special reference to the nuclear phenomena. Univ. Calif. Publ. Zool., 33:193-258. Turner, J.P., 1937. Cultivation of protozoa in Galtsoff et _al.: Culture methods for invertebrate animals, pp. 59-61* Uspenski, E.E. and W.J. Uspenskaja, 1925« Reinkultur und ungeschlechtliche Fortpflanzung des Volvox minor u n d Volvox globator in einer synthetischen Nährlösung. Z. Bot., 17:273-308. Watson, J.M., 1946. The bionomics of coprophilic protozoa. Biol. Rev., 21:121-139. Wells, M.M. and D.L. Gamble, 1928. Protozoan cultures, 2nd ed. Chicago: General Biological Supply House. von Wettstein, P., 1921. Zur Bedeutung und Technik der Reinkultur für Systematik und Floristik der Algen. Öst. bot. Z., 70: 23-29. Wichterman, R., 1949. The collection, cultivation, and sterilization of Paramecium. Proc. Penn. Acad. Sei., 23:151-180. Wilber, e.G., 1942. The cytology of Pelomyxa carolinensis. Trans. Amer. micr. Soc., 61:227-235Wilson, C.W., 1916. On the life-history of a soil amoeba. Univ. Calif. Publ. Zool., 16:241-292.

ADDENDUM SOIL-CHEESE MEDIUM This is useful for enrichment culture to develop larger populations of various protozoa obtained from the field, as well as for cultivation. Beilar ( 1 9 2 8 ) recommended preparation of tubes of medium by placing a piece of cheese or casein in a test tube, covering with a 4 cm.

layer of earth, then a layer of sand and water to a depth of 4 cm. over the sand. Sterilize, if desired, before adding sterile water. Small beakers, with a piece of cheese in the bottom covered with a layer of earth and a layer of sand, sterilized and filled to a depth above the sand with sterile water, covered with half of a small Petri dish, m a y be used.

II. COLLECTION AND CULTIVATION METHODS FOR SYMBIOTIC PROTOZOA

t i s s u e s of fish, a n d some o c c u r i n a m p h i b i a a n d reptiles. A m o n g the h o s t s are N o r t h A m e r i c a n smelt and sticklebacks. M i c r o s p o r i d i a a r e imAGAR-SERUM MEDIA, semi-solid. p o r t a n t i n insect p a t h o l o g y . M o r e t h a n a h u n G l a s e r and C o r i a (1935a) u s e d f o r culture of d r e d species h a v e b e e n d e s c r i b e d f r o m i n s e c t s ; B a l a n t i d i u m coli a m e d i u m c o n s i s t i n g of m o d i f i e d m o s t of t h e m b e l o n g i n the g e n e r a N o s e m a a n d Other important genera are PlistoR i n g e r s o l u t i o n (sodium c h l o r i d e , 6 . 5 g r a m s ; p o - T h e l o h a n i a . p h o r a , S t e m p e l l i a , a n d Perezia. L a r v a e of m o s t a s s i u m c h l o r i d e , 0.l4 gram; calcium c h l o r i d e , 0 . 1 2 g r a m ; s o d i u m b i c a r b o n a t e , 0.20 gram; s o d i u m q u i t o e s are c o n v e n i e n t s o u r c e s of m a t e r i a l s ; t h e y m a y c o n t a i n species of the f i r s t f o u r p h o s p h a t e , m o n o b a s i c , 0.01 g r a m ; w a t e r 1000 cc.) 250 cc. w a r m e d to 5 0 ° C , 2$ n u t r i e n t a g a r 25 cc., g e n e r a n a m e d a b o v e , b u t e s p e c i a l l y T h e l o h a n i a . r i c e s t a r c h 1 g r a m , f r e s h r a b b i t serum 12.5 cc., I n f e c t e d larvae a p p e a r m o r e o r less o p a q u e . M i c r o s p o r i d i a o c c u r i n the c o p e p o d C y c l o p s a n d pH a d j u s t e d at 7 . 2 to 7 . 4 , t u b e d in 15 cc. i n G a m m a r u s , among o t h e r a r t h r o p o d s . amounts. The stage of M i c r o s p o r i d i a m o s t r e a d i l y o b A d l e r a n d F o n e r (l94l) o b t a i n e d g r o w t h of Other i n t e s t i n a l a m o e b a e , f l a g e l l a t e s , a n d ciliates i n t a i n e d in h o s t e x a m i n a t i o n is the s p o r e . stages m u s t be s t u d i e d in s e c t i o n s o r t i s s u e a m e d i u m of 1 part 3$ a g a r , 8 parts L o c k e s o l u smears. t i o n (with 0.1$ d e x t r o s e ) , a n d 1 part i n a c t i ACTINOMYXIDIA. T h e m e m b e r s of t h i s small v a t e d s e r u m (horse, cow, g o a t , or m a n ) . A loopg r o u p of A m o e b o s p o r i d i a o c c u r e n t i r e l y in a n n e ful of rice s t a r c h is a d d e d to a tube. It w a s lids, o c c u r r i n g in the i n t e s t i n a l e p i t h e l i u m of a d v a n t a g e o u s to i n o c u l a t e the sterile m e d i u m Tubifex. w i t h S e r r a t i a m a r c e s c e n s (Bacterium prodigiosum) a f e w d a y s before i n o c u l a t i o n w i t h the p r o t o z o a . H A P L 0 S P 0 R I D I A are o r g a n i s m s of u n c e r t a i n systematic position. They occur in the cells, t i s s u e s , or cavities of c e r t a i n i n v e r t e b r a t e s a n d i n the g i l l s a n d organs of fish. The e a s i A M 0 E B 0 S P0RIDIA est f o r m to o b t a i n a n d s t u d y is C o e l o s p o r i d i u m p e r i p l a n e t a e , w h i c h o c c u r s in the l u m e n of the M Y X O S P O R I D I A are c o m m o n l y e n c o u n t e r e d a n d M a l p i g h i a n t u b u l e s of c o c k r o a c h e s . w i d e l y d i s t r i b u t e d p a r a s i t e s of f i s h , w h i c h are b y far t h e m o s t c h a r a c t e r i s t i c h o s t s . Some o c S A R C O S P 0 R I D I A c o n s i s t c h i e f l y of the g e n u s cur in c e r t a i n a m p h i b i a and r e p t i l e s . The m o s t S a r c o c y s t i s , of w h i c h s p e c i e s o c c u r p r i m a r i l y i n f r e q u e n t l y i n f e c t e d o r g a n is the gall b l a d d e r , the m u s c l e s of m a m m a l s a n d b i r d s . There are i n w h i c h the parasites o c c u r I n the l u m e n , a n d some in r e p t i l e s . C y s t s of S. t e n e l l a are o f t e n g e n e r a l l y b e l o n g to the g e n e r a M y x i d i u m , C e r a f o u n d i n the o e s o p h a g u s of s h e e p , a n d can b e o b t o m y x a , L e p t o t h e c a , C h l o r o m y x u m , S p h a e r o m y x a , or t a i n e d f r o m s l a u g h t e r h o u s e s . The organism can Zschokkella. C o e l o z o i c M y x o s p o r i d i a are also b e m a i n t a i n e d in m i c e i n f e c t e d b y f e e d i n g these f r e q u e n t l y f o u n d in the u r i n a r y b l a d d e r a n d cysts. S a r c o c y s t i s can b e s t b e s t u d i e d i n s e c u r i n i f e r o u s tubules of the k i d n e y . Other M y x o tions of the i n f e c t e d m u s c l e . s p o r i d i a , e s p e c i a l l y in the g e n e r a M y x o b o l u s , M y x o s o m a , a n d H e n n e g u y a , are h i s t o z o i c a n d i n h a b i t t i s s u e s of the i n t e g u m e n t or v a r i o u s i n AM0EB0Z0A t e r n a l o r g a n s of fish. T h e s e parasites m a y p r o duce w h i t i s h cysts or t u m o r - l i k e structures that T h e o n l y r h i z o p o d s l i v i n g in a n i m a l h o s t s a p p e a r o n the g i l l s , b o d y s u r f a c e , or f i n s of b e l o n g to the G y m n a m o e b a e a , the n o n - s h e l l e d infected fish. Amoebozoa. They are almost entirely inhabitants M y x o s p o r i d i a m a y b e o b t a i n e d b y a b s t r a c t i n g of the p o s t e r i o r a l i m e n t a r y t r a c t , t h o u g h a few m a t e r i a l f r o m the t i s s u e lesions or b y e x a m i n i n g o c c u r i n the m o u t h s of t h e i r h o s t s . Endamoeba the c o n t e n t s and scrapings of the gall b l a d d e r b l a t t a e is c o m m o n i n the i n t e s t i n e of c o c k or u r i n a r y b l a d d e r . roaches. S p e c i e s of E n t a m o e b a f r e q u e n t l y o c c u r i n the c a e c u m of r o d e n t s ; the b e s t k n o w n is M I C R O S P 0 R I D I A o c c u r also in fish, a m p h i b i a Entamoeba muris. E n t a m o e b a r a n a r u m o c c u r s in a n d r e p t i l e s and, u n l i k e M y x o s p o r i d i a , t h e y a m p h i b i a , a n d E. i n v a d e n s a n d E . t e r r a p i n a e i n p a r a s i t i z e i n v e r t e b r a t e s as w e l l . Hosts e x i s t M a n is the h o s t of three s p e c i e s of a m o n g i n v e r t e b r a t e s of v a r i o u s g r o u p s , i n c l u d i n g r e p t i l e s . p r o t o z o a , b u t t h e y are e s p e c i a l l y c h a r a c t e r i s t i c E n t a m o e b a , E . coli, E. h i s t o l y t i c a a n d E. g i n g i valis. E n d o l i m a x n a n a is a c o m m o n a m o e b o z o a n of of a r t h r o p o d s . M o s t species are i n t r a c e l l u l a r i n one or a n o t h e r of a v a r i e t y of tissues of the m a n , a n d species of the g e n u s o c c u r in s e v e r a l invertebrates and vertebrates. Iodamoeba body. A f e w m a y inhabit v a r i o u s tissues. The g e n u s G l u g e a contains species that form cysts in b ü t s c h l i i o c c u r s in m a n , m o n k e y s , a n d pigs. MATERIALS AND

PROCEDURE

[18]

MATERIALS AND METHODS IN THE STUDY OF PROTOZOA Amoebae m a y be obtained directly b y opening the bodies of the hosts, or m a y be found i n the feces. Trophic forms are not normally passed out of the body, but m a y be, in diarrhoeic conditions or after use of purgatives. Most of the symbiotic amoebozoa form cysts, and these are the forms generally found in the feces. For laboratory study of endozoic amoebae, trophic forms of Entamoeba m a y be found in the caecum of rats and other rodents. E. histolytica and other amoebae of m a n m a y be available in culture. Entamoeba invadens from reptiles is similar in structure to E. histolytica, grows readily in culture at room temperature and is active at room temperature, and can easily be obtained in all stages of its life history in culture. Cysts of amoebae m a y be found in feces of man, monkeys and other animals; and m a y be studied in culture, especially of reptilian Entamoeba. If cockroaches infected with Endamoeba blattae are available, trophies and cysts m a y be studied. Such a study will at once make it apparent that this, the type species of Endamoeba, is generically different from the amoebae of vertebrates placed in the genus Entamoeba.

BLOOD-AGAR MEDIUM N. N. medium is suitable for several species of trypanosomes, for Leishmania, and for Herpetomonas and other Trypanosomatidae. This m e d i u m consists of nutrient agar to which d e fibrinated rabbit b l o o d is added. The nutrient agar is made b y adding peptone, sodium chloride, and agar to meat extract. Meat extract m a y be prepared b y infusing 500 grams of lean ground meat in 1000 cc. of water for several hours at room temperature, straining, heating the mixture to boiling, and filtering. It m a y instead be made by using Bacto-Beef (a powder prepared from fresh lean beef, obtainable from the Difco Laboratories). 50 grams of the powder are added to 1000 cc. distilled water, infused at 50° C . for an hour, heated to 80° C. for 5 minutes, cooled and filtered. To prepare nutrient agar, add to the m e a t extract 2$ peptone (Bacto-Peptone or Difco N e o peptone), 0.5$ sodium chloride, and 2$ agar. The pH should be adjusted to about 7 A for w h i c h N/l sodium hydroxide m a y be used. The mixture is distributed in test tubes or small flasks and kept in a refrigerator, protected b y capping from evaporation if it is to be stored for long. To prepare blood-agar, the nutrient agar is m e l t e d in a water bath, cooled to 55° C., and from 10$ to 50$ of fresh defibrinated rabbit b l o o d added. After mixing, the tubes are cooled in a suitable position to form slants. N. N. N. medium is prepared without the use of meat extract and peptone. To 900 cc. distilled water are added 14 grams of agar and 6 grams of sodium chloride. The fluid is brought to the boiling point to dissolve the agar, dis-

19

tributed into tubes and autoclaved. Before starting a culture, one-third its volume of d e fibrinated rabbit b l o o d is added to the m e l t e d agar, and slants are formed. Rabbit b l o o d m a y be replaced by blood of m a n , cattle, sheep, guinea pig, o r rat; but horse blood is not satisfactory. (Berrebi, 1936.) Cultures m a y be kept at about 20° to 25° C., and subcultures made every ten days if maximum growth is desired. M a n y cultures will remain viable without transfer, if protected from evaporation by capping, for months or even years. Kelser's m e d i u m for Trypanosoma cruzi consists of nutrient agar to w h i c h dextrose and defibrinated guinea pig b l o o d are added. The n u trient agar is prepared b y adding 25 grams of Bacto-Beef to 500 cc. distilled water, heating in a w a t e r bath at 55° C. for a n hour, adding 12.5 grams Bacto-Peptone and 3 - 5 grams sodium chloride, heating w a t e r bath to boiling and leaving about 5 minutes for solution of peptone and salt. The b r o t h is then filtered through cotton and adjusted to neutral b y N/l sodium hydroxide. One per cent agar is then added and dissolved, the m e d i u m distributed in test tubes or small Erlenmeyer flasks, and autoclaved. To prepare for use, the agar is melted, cooled to 50° to 55° C., and addition made of 5$ of a 1$ dextrose solution and 5$ of defibrinated guinea pig blood. Details regarding maintenance of the cultures are g i v e n in the original publication (Kelser, 1936) and b y Craig (19^8).

BLOOD-RINGER MEDIUM To 1 cc. of Ringer solution (made w i t h 0.6$ sodium chloride) add 1 cc. of citrated h u m a n b l o o d (0.5 cc. blood and 0.5 cc. of 2$ sodium citrate). A tapered centrifuge tube m a y be used. K e e p the tubed m e d i u m i n the refrigerator two o r three days before using. K e e p cultures at 24°C., and transfer every two weeks or so (they sometimes last four weeks). In culture of certain pathogenic trypanosomes, it has b e e n found advantageous to add a little cholesterol (0.5 gms. per liter). F o r culture of Trypanosoma cruzi, a little dextrose should be added to the Ringer solution (5$ of a 1$ sugar solution). (Reichenow, 193^; Archetti, 1938.)

BLOOD-SERUM M E D I U M Nutrient solution is prepared as for B L O O D AGAR (Bacto-Beef infusion 5$, peptone 2$, sodium chloride 0.5$> pH 7-2 to J A , autoclaved). Rabbit serum (or h u m a n serum) is added in the amount of 10$ to 15$. CILIATA Opalinid ciliates can readily be obtained from frogs and toads. The genus most often encountered in A n u r a of this region is Opalina.

MATERIALS AND METHODS IN THE STUDY OF PROTOZOA Sources of holotrich ciliates are the mantle cavity of mussels, In which species of Conchophthirus, Ancistruma, and other thigmotrichs occur; the intestine of terrestrial and freshwater oligochaetes, where various genera of astomatous ciliates are to "be found; and the caecum and colon of horses, which contain a variety of rather specialized genera. The holotrichs Isotricha and Dasytricha occur in the rumen of certain ruminants, including cattle. The integument of fish is sometimes parasitized by Ichthyophthirius. The most widely distributed endozoic spirotrichs are Balantidium and Nyctotherus. Balantidium coli is common in the intestines of pigs and B. caviae in the caecum of guinea pigs. Amphibia sometimes harbor B. entozoon. Species of Nyctotherus occur in various arthropods and in amphibia. Common species are N. ovalis of cockroaches and N. cordiformis of frogs and toad3. The rumen of cattle, sheep and other ruminants is a consistent source of the diverse and specialized spirotrich ciliates of the family Ophryoscolecidae. The caecum and colon of the horse similarly regularly harbor several genera of the remarkable ciliates of the family Cycloposthiidae. Many peritrichs and suctoria occur attached to the outer surface of various invertebrates. Peritrichs often occur on aquatic insects and plankton cru3tacea; and suctoria are found on gammarid crustacea, hydroids, copepods, insects and other invertebrates. Non-stalked peritrichs, of the genus Trichodina and related forms, occur on the surface, and sometimes in open cavities, of many invertebrates and fish. Trichodina pediculus is frequently found on Hydra, often in company with the hypotrich Kerona pediculus. A special group of ciliates are the chonotrichs, most of which occur on marine crustacea, especially gammarids.

20

sodium chloride, 0.1$ sodium bicarbonate, 0.1$ potassium phosphate, monobasic, 0.01$ anhydrous magnesium sulfate, 0.01$ anhydrous calcium chloride. Powdered cellulose was prepared by treating absorbent cotton several days with strong hydrochloric acid. The inorganic medium is placed in a graduated cylinder in a water bath at 38° C. A gas mixture of 95$ nitrogen and 5$ carbon dioxide is bubbled through it for 15 minutes. 20 cc. of the fluid is placed in a 50 cc. Erlenmeyer flask with 16 milligrams of powdered cellulose and the same amount of powdered grass. 20 cc. of a vigorous culture is added. For the first inoculation 0.5 cc. of rumen contents is added to 30 cc. of fluid. Oxygen is displaced by bubbling the nitrogen carbon dioxide mixture through the culture, after which it is stoppered and incubated at 38° C. Cultures were transferred at 2-day intervals. Transfer is necessary at least when the grass and cellulose have collected near the surface, instead of being largely on the bottom as at first. The culture should be vigorously rotated to distribute the protozoa evenly before mixing 20 cc. of the culture with the fresh medium.

EGG SLANTS Wash 4 fresh eggs, break them into a flask with glass beads, add 50 cc. Ringer solution, Locke solution, or 0.85$ sodium chloride, emulsify, and tube in 3 to 5 cc. amounts. For coagulation and sterilization of slants, place tubes with the medium for the slants in a properly slanted position in the autoclave, closing all valves. Admit steam as quickly as possible to 15 pounds pressure. At the end of 10 minutes, open the valves so as to allow replacement of the air-steam mixture by pure steam, maintaining a constant pressure. Sterilize 20 minutes, close all valves, and allow the pressure to drop slowly.

CILIATE CULTIVATION Balantidium coli has been maintained in a medium consisting of one part of human serum and sixteen parts of Locke, Ringer, or sodium chloride solution, sterilized by filtration, inoculated by placing at the bottom about 0.1 cc. of intestinal contents with the ciliates. The FECAL EXTRACT MEDIUM will maintain Balantidium of the pig without the need of so frequent transfers. Semi-solid AGAR-SERUM MEDIA have also been used successfully. Nyctotherus ovalis of the cockroach has been cultivated in 5$ rabbit or human serum in 0.5$ sodium chloride. Nelson (19^3) cultivated Nyctotherus cordiformis in a medium based on alcoholic extract of various tissues. Hungate (1942) cultivated Eudiplodinium neglectum for 22 months in a medium with grass and cellulose maintained under anaerobic conditions . The inorganic solution consisted of 0.6$

EGG YOLK INFUSION MEDIUM Two or four eggs are hard boiled for 15 minutes, according to the concentration of medium desired. The yolks are crumbled in a beaker of 125 cc. of 0.8$ sodium chloride. The mixture is boiled 10 minutes, and the evaporated water replaced; then it is filtered by suction and restored to 125 cc. volume. The filtrate is autoclaved and the yolk precipitate filtered out. 125 cc. of M/l5 phosphate buffer (pH 7.5) is added. The medium may then be tubed in 5 cc. amounts, autoclaved, and stored in a refrigerator. Before introduction of amoebae, a loopful of sterile rice starch is added to a tube. Amoebae may persist for more than 9 days. (Balamuth and Sandza, 1944.) Dried egg yolk may be used instead of fresh egg yolk. Hitchcock and Rawson (1946) added Difco Bacto Coagulated Egg Yolk in amounts of

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA 1.6$ for fair growth (but longer survival) and 2 A and 3-2$ for a richer growth of amoebae. The medium is prepared b y adding 12, 18, or 25 grams of egg yolk to 375 cc. of sodium chloride, letting the mixture stand 10 minutes, boiling 10 minutes, filtering, and restoring to 375 cc. 375 cc. of M/15 NaoPO^.12 H 2 0 (pH 7.5) is added. The m e d i u m m a y be tubed in 5 cc. amounts, autoclaved 20 minutes, and stored, with rice powder added before inoculation. Balamuth (19^6) gave the following directions for preparing the medium from dried egg yolk., A n amount of 36 grams of dehydrated egg yolk is m i x e d w i t h an equal amount of distilled water, and added to 125 cc. of 0.8$ sodium chloride solution. After thorough mixing, the preparation is boiled for 10 minutes, and water is added to restore the volume of fluid. After filtering, (through cloth or cotton), the fluid is restored to the original volume with 0.8$ sodium chloride. There is added an equal amount of M / 1 5 potassium phosphate buffer pH 7.5' The m e d i u m m a y be tubed i n 5 cc. amounts, autoclaved, and stored if desired. A small amount of sterile rice powder is added before inoculation. Half the amount of yolk m a y be satisfactory for ordinary purposes. If it is desired to provide a richer medium, there is added one part in ten of a nutrient solution prepared b y heating 5$ in water of p o w dered W i l s o n liver concentrate or Lilly's liver extract and filtering off the sediment. F E C A L EXTRACT MEDIUM Balantidlum coll was cultivated b y Nelson, 19^0 in a m e d i u m prepared from the caecal contents of the pig; this m e d i u m should also be useful for other ciliates of the large intestine, preparing it from intestinal contents of the host. One part of caecal contents is m i x e d w i t h 9 parts of Ringer solution, and the mixture strained through a sieve and then through a funnel with a thick pad of absorbent cotton. If the medium, not autoclaved, is kept at room temperature or in a refrigerator, the pH will rise to 8.0 or more in about a month. A t a n initial pH of 8.0, w h e n inoculated, cultures of Balantidlum lived longest. A t the time of inoculation rice starch is added, and the cultures are kept at b o d y temperature. Addition of starch every 2 or 3 days m a y prolong the life of the culture. Transfers should be made every 1 to 3 weeks.

FLAGELLATA The largest number of symbiotic flagellates inhabit the lumen of the intestine of their hosts. Flagellates m a y occur in the blood of vertebrates, but for the most part only the one genus Trypanosoma is involved. Some flagellates occur in special sites, as the m o u t h and anteri-

21

o r alimentary tract, the reproductive tract, the b o d y cavity of certain invertebrates, and tissue cells of vertebrates. These are specialized habitats occupied b y a relatively small number of species. The number of epibiotic flagellates is not large, but some do occur o n aquatic invertebrates and vertebrates . Among the hosts from w h i c h symbiotic f l a gellates m a y readily be obtained for laboratory study are wild and laboratory rodents; lizards, salamanders and frogs; termites and cockroaches; m u s c o i d flies; bugs of the order Hemiptera, and other insects. The groups containing pigmented flagellates include few forms that are symbiotes; but there are some symbiotic euglenoid flagellates and dinoflagellates. The most frequently encountered euglenoid is Euglenamorpha, w h i c h occurs in the intestine of tadpoles. Certain species of Euglena or similar flagellates m a y be found attached to Cyclops or Daphnia in freshwater plankton. Colorless euglenoid flagellates of the Astasia-type (Khawkinea) are not infrequently encountered in freshwater Turbellaria, and m a y occur also in rotifers, gastrotrichs, Chaetogaster, and other hosts. Symbiotic dinoflagellates, especially Blastodinium and Syndinium, m a y be looked for i n marine copepods. Oodinium ocellatum is sometimes found o n the gills and skin of marine f i s h in aquaria (Nigrelli, 1936), and 0. limneticum has b e e n found o n fresh-water tropical fish (Jacobs, 19^6) in aquaria. The groups of non-pigmented Flagellata contain a large number of symbiotic forms. The Rhizomastigina are m o s t l y free-living flagellates, but one, M a s t i g i n a hylae, is fairly frequent in the intestine of tadpoles. On the skin and gills of freshwater fish one o f t e n finds the tetramitid Costia necatrix. Beetle grubs, mole cricket s, and crane f l y larvae contain small flagellates of the g e n e r a Monocercomonoides, Retortamonas, and in some Polymastix. The first two genera are fairly widespread in both insects and mammals. Monocercomonoides has species common in g u i n e a pigs and ground squirrels. Chilomastix is also a widespread intestinal flagellate, especially in vertebrates, but it occurs also in some invertebrates. Species of Chilomastix easily obtainable for study are C. caulleryi of amphibia, C. intestinalis of g u i n e a pigs, C. m a g n a of ground squirrels, C. bettencourti of rats, and C. mesnili of man. Endozoic bodonids and Trypanosomatidae inhabit b o t h invertebrates and vertebrates. Proteromonas occurs in the alimentary tract of lizards and salamanders, and K a r o t o m o r p h a in amphibia. Cryptobia helicis is f o u n d in the seminal receptacle of snails of the genus Helix. Flagellates related to it occur in the aliment a r y tract and b l o o d of fish. Flagellates of the family Trypanosomatidae occur m o s t l y in the lumen of the alimentary tract of invertebrates and in the b l o o d and

MATERIALS AND METHODS IN THE STUDY OF PROTOZOA tissue of vertebrates. Certain flagellates of the leptomonad type (Phytomonas) inhabit the latex of plants of the families Euphorbiaceae and Asclepiadaceae, as well as certain other lactiferous plants. A readily obtainable form for laboratory study is Herpetomonas muscarum, which occurs commonly in the gut of muscoid flies. Crithidia is common in the intestine of a variety of Hemiptera. The box-elder bug, Leptocoris trivittatus, often contains Crithidia leptocoridis. Trypanosoma may be found in natural infection in various vertebrates, including rats and mice, various wild rodents, frogs and salamanders, and fish. For laboratory study certain pathogenic trypanosomes (as T. equiperdum, T. brucei), which develop massive infections in laboratory rodents, may be used. The genus Hexamitus (including Octomitus) contains species some of which are free-living In fresh or salt water, others symbiotic in aquatic and terrestrial animals. H. intestinalis and other species may be found in salamanders and frogs. H. muris and H. pulcher are present in rats, mice and other rodents. H. meleagridis in the small intestine of gallinaceous birds has been considered responsible for enteritis. H. salmonis sometimes is considered a source of trouble in fish hatcheries. Species of Giardia inhabit the small intestine. The most readily obtainable forms are G. muris of rats and mice, G. duodenalis of rabbits, G. lamblia of man, and G. agilis which occurs in tadpoles. Trichomonad flagellates are a large group consisting of several families. The family Trichomonadidae is widespread in invertebrates and vertebrates. The genus Tritrichomonas includes the species T. augusta and T. batrachorum, common in amphibia; T. muris abundant in rats, mice and other rodents; and T. cavlae of guinea pigs. The genus Trichomonas includes T. gallinae, not infrequent in the throat of pigeons and other gallinaceous birds; T. tenax, fairly common in the mouth of man; and T. vaginalis of the vagina of man. Pentatrichomonas hominis, from the intestine of man, cats, dogs and other hosts, can easily be maintained in culture; Trichomonad flagellates have developed in great abundance and variety in the intestine of termites. A large and useful form for study is Trichomonas termopsidis, in termites of the genus Zootermopsis. Devescovinid flagellates occur in most termites of the family Kalotermitidae. The flagellate family Calonymphidae, including the genera Coronympha, Metacoronympha, Stephanonympha, and Calonympha, is also represented in many termites of that family. Hypermastigote flagellates occur only in termites and cockroaches. Suitable forms for study are found in termites of the genus Reticulitermes. Species of Trichonympha occur in Reticulitermes (T. agilis) and in Zootermopsis (T. campanula, T. collaris, and T. sphaerica). The hypermastigotes in cockroaches are Lophomonas blattarum and L. striata; and a variety of forms, including several species of Trichonympha, In Cryptocercus punctulatus.

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FLUID MEDIA Egg and serum slants are often used in cultivation of intestinal amoebae and flagellates, but growth of many forms proceeds satisfactorily in simple, strictly fluid media, to which rice starch may be added or not. 1. EGG YOLK INFUSION 2. Buffered saline serum. (Pavlova, 1938.) Sodium chloride 0.9$, 9 cc.; horse serum, 0.5 cc.; S^rensen phosphate buffer pH 6.5, 2 cc. Buffer solution is added each day. 3. Locke-egg medium. (Hogue.) One hen's egg is shaken with glass beads in a flask, 200 cc. Locke solution added, mixture heated and agitated over water bath 15 minutes, filtered through cotton with suction, tubed In 6 cc. amounts, autoclaved. 4. Serum fluid media. (Craig, 1926; Jirovec and Rodova, 1940.) Locke solution with human, horse, or rabbit serum 1:7, sterilized by filtration. 5$ serum, preferably human or horse serum, in modified Ringer solution or in 0.6$ sodium chloride solution. Can be sterilized by heat. Addition of rice starch gives more luxuriant growth. 5. Dried blood serum and gastric mucin medium. Wenrich (1946, 1947a,b) cultivated trichomonads and some other intestinal flagellates in tubes with about 10 cc. of modified RINGER SOLUTION, Drbohlav formula, containing 0.2$ gastric mucin or Loeffler's dried blood serum. From time to time, when populations begin to decline, mucin or blood serum is added dry or, if evaporation of fluid necessitates replenishment, nutrients may be dissolved in distilled water. Mucin and serum may be added alternately every 2-4 days. By this method of feeding and replenishment of cultures, the flagellates were maintained in the original tubes for weeks or months, or even years. Tritrichomonas batrachorum lived in an original tube for four years. HIST0M0NAS MELEAGRIDIS This flagellate may occur in turkeys, in which it is the cause of blackhead disease, and has also been found in chickens, grouse, quail, and pheasants. It lives in the lumen of the caeca and in lesions in the liver. De Volt (1943) cultivated Histomonas in a medium of turkey or chicken serum in Locke solution, with rice starch added. To the Locke solution (water 1000 cc., sodium chloride 9 grams, calcium chloride and sodium bicarbonate 0.2 gram each, potassium chloride 0.4 gram, glucose 2 grams) two percent of fresh turkey or chicken serum and two percent N/20 sodium hydroxide are added and the medium autoclaved. A little sterilized rice starch is added to each culture tube. If bacteria become too numerous glucose is omitted and half the amount of serum used. Transfers are made twice a week.

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA HOST EXAMINATION

23

hoppers and mole-crickets; ground-dwelling and some wood-boring beetle larvae, mealworms and Endozoic protozoa m a y inhabit open cavities other Coleóptera; larvae of crane flies and of their hosts or blood or tissue. Open cavicaddis worms; muscoid flies and other Diptera. ties are those that lead by openings to the e x Histozoic sporozoa and microsporidia are freterior, as the alimentary tract, the mantle quent i n m a n y groups of insects. cavity of molluscs, and the reproductive tract. Earthworms and other oligochaete annelids The gall bladder and urinary bladder, which can profitably be examined, especially for monooften are habitats of certain protozoa in fish cystids and intestinal ciliates. If fresh-water and amphibia, m a y also be placed in this catemussels are available, the mantle cavity is gory. The blood of vertebrates is a frequent likely to contain certain ciliates. habitat, and protozoan forms m a y often be found If the collector has access to the seain the haemocoele of arthropods. Tissueshore, examination m a y be m a d e of the intestine inhabiting forms occur in cells and tissues of of sea urchins; the cloaca and respiratory trees m a n y parts of the body : the cellular lining of of holothurians; the gill or mantle region of the intestine, the fat body of insect larvae, mussels, clams, limpets, chitons and other m o l the musculature, the r e t i c u l o e n d o t h e l i a l system, luscs; the intestine and tissues of polychaete and other tissues. The same protozoan may, in worms; the surface of hydroids; the appendages, different stages, inhabit tissues and blood of gills, and alimentary tract of amphipods, isoone host and the lumen of the alimentary tract pods, and crabs; the b o d y cavity of amphipods of another. Intestinal coccidia, developing in such as sand hoppers; and of possible habitats the lining of the intestine, produce oocysts in other invertebrates. There is scarcely any that occur in the lumen, and so come within that marine invertebrate that does not m e r i t examio p e n cavity category of examination. nation either for attached forms or interior symbiotes. Parts of the alimentary tract of some animals are almost certain sources of protozoa. F o r a complete examination of every vertePopulations of ciliates are present in the rumen brate, from fish to mammals, the blood should be and reticulum .of almost all normally fed cattle, observed and blood films made. Trypanosomatidae sheep and other ruminants. Ciliates occur regu- and m a l a r i a parasites can best be kept available larly, in variety and abundance, In the caecum for laboratory study in infections maintained in of horses. The caecum of rodents such as labolaboratory animals. ratory rats, mice, and guinea pigs, wild rats In fish, one m a y examine white spots and and m i c e , ground squirrels and others often con- tumors o n the skin and gills; look at the contains a good population of protozoa. Those same tents of the gall bladder and urinary bladder, animals sometimes have flagellates in the small and possibly the swim bladder; make scrapings or intestine. Frogs and toads usually harbor certissue samples from the skin and gills, as well tain flagellates and ciliates in the intestine as investigating the contents of the alimentary and cloaca. A n outstanding instance of consistract and the blood. tency of occurrence of protozoa i n invertebrates is found in termites. Every individual in the families of lower termites normally contains a INTESTINAL FLAGELLATES A N D AMOEBAE rich fauna of flagellates. Cockroaches are often hosts of protozoa. Examination of the aliA summary of various cultivation methods m e n t a r y tract of pigs, monkeys, man, and chickfor symbiotic amoebae and flagellates is g i v e n ens will often be productive. Material to g a i n b y K o f o i d and McNeil (1937) and b y Craig (19^8) acquaintance with various types of protozoa inin his book on the laboratory diagnosis of prohabiting the alimentary tract can be obtained tozoan diseases. Use of EGG SLANTS, SERUM without difficulty from m a n y generally available SLANTS or LIVER INFUSION AGAR, w i t h LIQUID MEDIA animals. TO COVER SLANTS, is advantageous for m a n y purposes but is not essential for cultivation of One who has access to zoological gardens m a n y of the protozoa. EGG Y O L K INFUSION MEDIUM will be able to obtain m u c h material from the and other F L U I D MEDIA provide simpler culture animals there. Autopsy m a y sometimes be possible. A n opportunity to obtain ciliates from the methods that m a y be adequate for ordinary purposes. Addition of STARCH, of w h i c h the grains alimentary tract of elephants, giraffes, or are small enough for ingestion, aids growth of camels should be seized. I n m a n y instances one m a n y forms. See also TRICHOMONAD FLAGELLATES. can secure protozoa In cultures inoculated from feces. The non-carnivorous mammals and the repA m e d i u m based o n alcoholic extract of tiles are likely to be especially productive. tissue or egg yolk has been u s e d successfully Among arthropods are m a n y that contain pro- for Entamoeba histolytica (Nelson, 19^7). Whole tozoa passed from individual to Individual of a liver, or egg yolk, is placed in nine times its species b y contaminating forms, and some that volume of 95$ alcohol. After 48 hours, during are hosts for stages of parasites of vertebrates w h i c h It is shaken at intervals, it is ready for w i t h w h i c h they become infected i n ingesting use, and will last indefinitely. Clear superblood. Besides cockroaches and termites, frenatant f l u i d is drawn off b y a pipette as needed. quent hosts of various species are centipedes To prepare cultures, 10 cc. of the f l u i d is placed in a flask and heated to drive off the and millipedes; other Orthoptera, as grass-

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA alcohol. 20 oc. of 2$ agar in buffered 0.5$ salt solution is added. The solution is tubed in 2 cc. quantities, slants made, and these covered with 0.5$ salt solution. Growth is best at pH 7.4 - 7-6. Rice starch is added before inoculation. Infections with certain intestinal protozoa, especially trichomonads, from man and other warm-blooded animals, can be established in parasite-free chicks about four days old. Inoculation of chicks is said to be a practicable method of increasing the numbers of certain forms and maintaining them for laboratory study. (See Hegner, 1929a, 1929b. 1937.) ISOLATION PROM BACTERIA Washing and migration methods have been used to free certain symbiotic protozoa from bacteria, but some have found them uncertain in results. Stone and Reynolds (1939) obtained bacteria-free Pentatrichomonas hominis by a method of migration in a liquid medium (Ringer and horse serum, 8:1). A length of 6 mm. glass tube is drawn out so as to produce a long capillary tube, with a portion of the original-sized tube at one end. The capillary tube is bent in a series of loops, which serve as traps for bacteria. By means of a syringe attached to the broad end, medium is drawn up to fill the tube and the capillary terminus is sealed. The apparatus is placed in a vertical position, tested for sterility, and trichomonads are introduced into the broader end. Flagellates free of bacteria may have reached the bottom of the capillary tube by 2 days or more, whereupon sections of tube can be removed and placed with sterile precautions into culture medium. It is possible to make microscopic observations on the migrating trichomonads in the capillary tube. Antibiotics have been used to obtain bacteria-free cultures. Johnson, Trussell, and Jahn (194-5) obtained bacteria-free strains of Trichomonas vaginalis by introducing vaginal discharge into culture tubes containing 5>00010,000 units of penicillin in 10 cc. of C.P.L.M. medium, and incubating 60 hours before transfer. Mahmoud (1945) used penicillin to isolate T. foetus in bacteria-free cultures; and Morgan (1946) eliminated various species of bacteria from cultures of T. foetus by making two transfers into medium containing 100-500 Oxford units of penicillin per cc. Seneca, Henderson, and Harvey (1949) described the use of antibiotics to purify contaminated cultures of Leishmania and Trypanosoma. An amount of 5000 units of penicillin and 5000 units of streptomycin is added to culture tubes in 5 oc. of salt solution.

LIQUID MEDIA TO COVER SLANTS 1. Horse serum diluted 1:6, 1:7, or 1:8 with Ringer or Locke solution. Human or rabbit serum may also be used.

24

2. Egg albumen diluted in Ringer, Locke, sodium chloride solution, or buffered solutions at pH 6.4 to 7.4. A 10$ solution of albumen may be used, or the whites of 4 eggs may be mixed in 1000 cc. of the fluid. 3. Loeffler's dehydrated blood serum solution prepared by adding 0.25 gram to 1000 cc. of Ringer solution. The mixture should be boiled one hour to facilitate solution, then sterilized in an autoclave. 4. Locke-Blood medium. (Kofoid and Wagner, 1925; Kofoid and McNeil, 1932.) The fluid used to cover slants consists of 2.5 oc. of fresh, defibrinated rabbit blood in 500 cc. of Locke solution.. 5 . Locke solution without addition of any substance except rice flour, and some growth may occur without the starch. (Reardon and Rees, 1939.) A loopful of rice starch may be added to any of these media before inoculation. Cultures in single tubes may be maintained for two weeks or so by pouring off the liquid each day and replacing with fresh fluid, adding each time a small amount of rice starch. (Deschiens, 1927-) LIVER INFUSION AGAR Liver infusion agar may be bought from the Difoo Laboratories as Bacto-Entamoeba medium. Directions for using it for E. histolytica are given in the Difco Manual. 33 grams are mixed in 1000 cc. distilled water, boiled for 1 to 2 minutes, tubed and sterilized in the autoclave. The tubes are slanted so that the fluid will solidify as a slant; they should be so placed as to obviate the formation of a butt. The slants are covered with sterile horse serum plus salt solution 1:6, or with the fluid of Craig, consisting of inactivated sterile horse serum, 1 part, and S^rensen phosphate buffer pH 7.4, 10 parts. A loopful of sterile rice flour or rice starch should be added before inoculation. (Cleveland and Sanders, 1930; Cleveland and Collier, 1930; Craig, G.M., 1936.) LOCKE SOLUTION Formula used in original Entamoeba-medium: Sodium chloride, 9 grams; calcium chloride, 0.2 gram; potassium chloride, 0.4 gram; sodium bicarbonate, 0.2 gram; dextrose, 2.5 grams; water 1000 cc. Modified (Stone, 1935.) Sodium chloride, 8 grams; calcium chloride, 0.2 gram; potassium chloride, 0.2 gram; magnesium chloride, 0.01 gram; sodium bicarbonate, 0.4 gram; sodium phosphate, dibasic, 2 grams; potassium phosphate, monobasic, 0.3 gram; water 1000 cc. LOPHOMONAS BLATTARUM Lorenc (1938) obtained growth of this flagellate from cockroaches in 0.8$ salt solution

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA with yeast added for food. The yeast was grown separately in lemon juice diluted with an equal amount of tap water, inoculated from the gut of a cockroach in w h i c h Lophomonas was present. To use the yeast for the culture, it was washed b y repeated centrifuging with water, suspended in salt solution, and added to salt solution w i t h Lophomonas in a vial or tube. The yeast should not be over-abundant. For later feeding, fluid m a y be removed from the upper part of the culture to maintain the volume as new fluid with yeast is added. OPALINID CILIATES Opalina from anurous amphibia has b e e n kept alive in a solution consisting of 0.8$ sodium chloride, 100 parts; 30$ potassium-sodium tartrate (Rochelle salt), 5 parts; distilled water, 400 parts. (Putter, 1905; Konsuloff, 1922.) Putter solution m a y be boiled and changed each day, or frequently. Too much oxygen content is detrimental to the ciliates. For nutriment Konsuloff added, at each change of fluid, a drop of fresh or boiled bouillon made of frog intestine contents in Putter solution, centrifuged; or a drop of egg albumen dilution in this solution, broken up and centrifuged. Cultures were maintained as long as 2 months. Lwoff (1948) freed Cepedea dimidiata, from Rana esculenta, from bacteria b y repeated wash-ing and obtained cultures in a medium (sodium chloride 4 grams; magnesium sulfate, crystal., 0.01 gram; potassium phosphate, monobasic, 1 gram; purified gum arabic, 10 grams; distilled water, 1 liter; adjusted to pH 7.1 with s o d i u m hydroxide) to w h i o h was added extract of frog liver, yeast extract, beef extract, peptone, and either cysteine hydrochloride or vitamin C. Preparation of the medium is described in Lwoff's paper. The ciliates were cultivated through 15 transfers, becoming as numerous as about 120 per cc. in the pure cultures. Bacteria appeared in certain cultures, and in these m i x e d cultures Cepedea developed m u c h better, becoming about 4 times as abundant and developing cysts. PRESERVATION BY LOW TEMPERATURE FREEZING Certain trypanosomes and Plasmodium species can be preserved alive for long periods b y quick freezing in d r y ice and alcohol, and maintaining low temperature by dry ice refrigeration. A simple cabinet for this purpose was described b y Manwell (1943). B l o o d in a thin-walled glass tube is placed in the alcohol, w h i c h has b e e n chilled b y d r y ice to - 5 5 ° C. to - 7 8 ° C., and the tube is rapidly rotated. It is then stoppered and kept partly immersed in the alcohol. For use, the blood is thawed and the animal which is to serve as a host is inoculated. Parasites of bird malaria have b e e n kept for about 6 weeks to 7 months in this way; T. equiperdum was preserved for 14 months. (Stone and Thompson, 1940; Manwell, 1943.)

25

RINGER SOLUTION Modified, Drbohlav formula. Sodium chloride, 6 grams; sodium bicarbonate, 0.1 gram; potassium chloride, 0.1 gram; calcium chloride, 0.1 gram; water, 1000 cc. The solution should be sterilized b y filtration, because of the presence of sodium bicarbonate . Warm-blooded, without sodium bicarbonate. Sodium chloride, 8.5 grams; potassium chloride, 0.25 gram; calcium chloride, 0.3 gram; water, 1000 cc. Cold-blooded. Sodium chloride, 6.5 grams; potassium chloride, 0.25 g r a m ; calcium chloride, 0.3 gram; sodium bicarbonate, 0.2 gram; water, 1000 cc.

SERUM SLANTS Slants m a y be prepared of inspissated horse serum, and covered w i t h serum-saline 1 to 6 or 1 to 8. Loeffler dehydrated beef serum m a y be used. The dried blood serum m a y be purchased. An amount of 80 grams is dissolved in 1000 cc. water at 42° to 45° C. The solution is coagulated and sterilized in a n autoclave or a n Arnold sterilizer. The slants m a y be covered b y fresh horse serum and saline 1 to 6, or by dilute albumen, or other FLUID MEDIA F O R COVERING SLANTS. (Cleveland and Sanders, 1930.)

SP0R0Z0A (See also AMOEBOSPORIDIA) The two m a i n divisions are C0CCIDI0M0RPHA and GREGARININA. Coccidiomorpha are parasites primarily of tissues and blood cells of v e r t e brate and invertebrate hosts. Gregarines occur in invertebrates. The Coccidiomorpha consist of two groups, the C0CCIDIA and the ADELEIDA. Of the three categories of Coccidia, the EIMERIIDEA are mostly parasites of the intestinal epithelium, seldom the bile duct epithelium. I n certain fish, species of Eimeria occur i n tissues of various organs including, besides the alimentary tract, the testes, liver, swim b l a d der, spleen, and kidneys. Lankesterella d e velops in endothelial cells of b l o o d vessels of frogs, and sporozoites enter red b l o o d cells. Eimeriidea, with few exceptions (Aggregatidae, Lankesterellidae), have one host only. In most, oocysts are found in the intestinal contents or feces of the host. They m a y be found i n mature development in certain internal organs of fish. The species of the other two categories of Coccidia, the HAEM0SP0RIDEA and PIROPLASMIDEA, have two hosts, a vertebrate and an invertebrate. I n the vertebrate the parasites occur in tissue cells and blood cells, and infection is u s u a l l y detected by b l o o d examination.

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA The second group of the Coccidiomorpha, the Adeleida, consists of two categories: the ADELEIDEA and the HAEMOGREGARINIDEA. Adeleidea have a coccidian type of oocyst and parasitize the intestinal epithelium or interior tissues of their hosts. There is only one host, w h i c h is a n invertebrate. The haemogregarines develop in Interior tissues and blood cells of vertebrates, and have two hosts in the life cycle, the second being a blood-sucking invertebrate. Cysts of Adeleidea m a y be found in the intestinal contents or feces, or in the bodies of the invertebrate hosts; or (Klossiella) in the urine or kidneys of guinea pigs or mice. Haemogregarines are found b y blood examination of vertebrates. The two divisions of the Gregarinina are the EUGREGARINIDA and SCHIZOGREGARINIDA. The members of the first group are, in general, intracellular for only part of the growing period, and later forms in development often occur in the lumen of the intestine, but in some in the b o d y cavity. The hosts are invertebrates of various phyla, especially arthropods. Schizogregarines occur in the intestine or tissues, and are found in various arthropods, annelids, and occasionally in tunicates. Sporozoa for laboratory study m a y be obtained from sources referred to i n what follows, as well as from other sources. Coccidia of the group Eimeriidea occur comm o n l y in certain birds and mammals. Rabbits are often infected with Eimeria stiedae in the liver and w i t h E. perforans and other species in the Intestine. Coccidia are often present in chickens; Eimeria tenella, E. necatrix, E. maxima, E. acervulina, E. mitis and other species m a y be found. Eimeria falciformis of mice is a useful form for laboratory study if an infection can be found. White mice can be infected, cysts kept moist remaining infective for a year or more. Developmental stages can be studied in pieces of the mucous membrane of the small intestine m o u n t e d on slides. Species of the genus Isospora m a y be obtained from cats and dogs. Aggregata is excellent material for laboratory study of developmental stages. It has two hosts, a cephalopod and a crab. In Europe, Sepia officinalis is almost always infected. On the Pacific Coast, similar stages to those in Sepia have b e e n found in Octopus. Schizogony occurs in the gut of crabs. Those stages are not so easily obtained as are gametogony and sporogony in the cephalopods. The Haemosporidia include the genus Plasmodium, of w h i c h the species from m a n are widely used for laboratory study. Species of avian Plasmodium can often be found in wild birds and maintained, according to the species, in canaries, ducks, and chickens. Plasmodium gallinaceum is subject to severe restrictions, but P. cathemerium, P. relictum, and P. lophurae are obtainable. Species of Haemoproteus and Leucocytozoon m a y be found in various birds; one of the former genus is not uncommon in quail in California. Babesia bigemina and Babesia canis are

26

found in certain areas in cattle and dogs. Usually for laboratory study of Babesia, as also for Theileria, one must depend on prepared blood films. Adeleid coccidiomorphs m a y be found in a v a r i e t y of invertebrates. Species of Adelina and related genera have b e e n reported from the M a l p i g h i a n tubules of fleas and aquatic beetles, the fat bodies of the wood roach Cryptocercus, the intestinal epithelium and salivary glands of Hemiptera, the gut epithelium of crane-fly larvae, and elsewhere. Adelina deronis was found in the lining of the coelom of a small aquatic annelid, Dero limosa. Species of K l o s s i a are often found in the kidneys of land snails, especially of the genus Helix. Haemogregarines w h i c h are likely to belong to the genus Karyolysus occur in snakes and lizards. Species of Hepatozoon are sometimes present in rats and dogs, and have been reported from birds. Productive sources of acephaline gregarines are earthworms; various species inhabit the Diplocystis seminal vesicles, some the coelom. occurs in the body cavity of cockroaches. Cephaline gregarines m a y be found in the intestine of m a n y insects, including cockroaches, earwigs, grasshoppers, mealworms, dermestid and other beetles, caddis worms, and others. Some of these can be maintained in the laboratory with infections. Coleoptera and Orthoptera are the insect orders most frequently parasitized b y gregarines. Gregarines also occur in some other invertebrates. Besides insects, arthropods that often contain gregarines are barnacles, crabs, amphipods, centipedes, and millipedes. A n easily controlled laboratory source of cephaline gregarines is Tenebrio molitor, the mealworm, in the intestine of the larva of w h i c h Gregarina cuneata and other species frequently occur. Gregarines m a y be found in the intestine and body cavity of cockroaches; Gregarina blattarum m a y be found in the intestine of Blatta orientalis, Periplaneta americana, and B l a t t e l l a germanica. Lumbricus terrestris and other earthworms can be kept in large boxes filled about twelve inches deep w i t h dead leaves and loam, or with light loamy soil. Unless there is m u c h organic material present, the worms should be fed every 2 or 3 weeks w i t h m o i s t bread crumbs or corn meal over w h i c h soil is spread. The loam should be kept moist but not wet. Mealworms (Tenebrio molitor and T. obscurus) m a y be reared in large containers in which wheat b r a n is placed to a depth of 6 or 8 inches, m i x e d with a little graham flour and commercial meat scrap. Raw, f r e s h carrots, potatoes, or lettuce should from time to time be placed o n top of the bran. The container m a y be covered w i t h a sheet of metal perforated b y small holes. Another m e t h o d of rearing mealworms is in large containers in w h i c h a layer of chick m a s h is placed to a depth of l/k inch, covered with b u r lap, and four o r five more thin layers of m a s h

MATERIALS A N D METHODS IN THE STUDY OF PROTOZOA and burlap placed over it. The culture is started b y placing a large number of worms in each box, and is sprinkled with water each day. The box should be uncovered or covered with mesh.' In old boxes, fresh layers of m a s h and burlap m a y be placed over the used-up layers. Occasionally pieces of potato or carrot m a y be added to feed adult beetles. Cockroaches m a y be kept in battery jars with a layer of sawdust and a small pan of water, the jar being covered with cheesecloth. They m a y be fed o n pieces of apple, or on a mixture of bread, cornstarch, and water. The supply of water must be kept up. Centipedes m a y be kept in Petri dishes floored with filter paper which is kept moist, and given termites or other suitable insects for food. Schizogregarines m a y be looked for in the Malpighian tubules of mealworms and other beetles, and in the fat bodies of the flour m o t h E p h e s t i a kuhniella and the wax m o t h Galleria mellonella. Stocks of these insects, in w h i c h infections are established, can be kept in the laboratory. Species of Selenidium m a y be found i n the intestine of polychaete worms.

27

TRICHOMONAS VAGINALIS

The flagellate was cultivated in S E R U M FLUID MEDIA b y Jirovec and Rodova (19^0). Powell (1936) u s e d dried blood serum and h u m a n serum. To RINGER SOLUTION, modified, add Q.2^% Loeffler's dried b l o o d serum and fresh human serum, with a loopful of rice starch to 8 to 10 cc. in a culture tube. It was g r o w n in 5$ h u m a n serum in m o d i f i e d Ringer, over LIVER INFUSION AGAR slants b y Johnson (1940). F o r bacteria-free cultures i n this medium it was found advantageous to adjust the initial pH to about 5 . 8 (with N/l sodium hydroxide or hydrochloric acid), to buffer w i t h 0.25$ sodium phosphate, and to use fluid made u p with 0.2$ dextrose and 0.1$ sodium thioglycolate. F o r bacteria-free Trichomonas vaginalis the C. P. L. M . m e d i u m (cysteine, peptone, liver, maltose) m e d i u m is recommended (Trussel and Johnson, 194-5)• The ingredients brought t o gether are: Bacto peptone, 32.0 grams; Bacto agar, 1.6 grams; cysteine monohydrochloride, 2.4 grams; maltose, 1 . 6 grams; Bacto liver infusion, 320 cc.; m o d i f i e d Ringer, 960 cc.; N/l sodium hydroxide, 11 to 13 cc. The m i x t u r e is heated in a water bath to m e l t the agar, f i l tered through coarse filter paper, methylene blue added (0.7 cc. of 5$ solution), the pH adSTARCH justed to 5 . 8 to 6.0 w i t h N/l sodium hydroxide or N/l hydrochloric acid. It is tubed in 8 cc. A large number of phagotrophic intestinal amounts and autoclaved. 2.cc. of sterile h u m a n protozoa have b e e n found to grow well w h e n starch is added to the medium. Except for large serum is added for culture, either before storprotozoa, the grains must be small for ingestion. ing or w h e n ready for use. Storage should be at room temperature. Subcultures are made every A loopful, m o r e or less, of rice starch m a y be other day b y transfer of about 0.05 cc. added to a tube of culture. Bacto-Rice powder can be purchased from the Difco Laboratories, or Chinese rice flour m a y be used. The 3tarch can be sterilized in a d r y test tube plugged w i t h TRITRICH0M0NAS FOETUS cotton, i n which the starch ia distributed with the tube horizontal, heated d r y at 90° C. for a n M o r g a n (1944) gave a summary of various hour in each of three periods separated b y a m e d i a used in cultivation of this flagellate of day. If over-heated the starch will turn brown. the reproductive tract of cattle, w i t h references to the original formulas. Among the simpler m e d i a used successfully TRICH0M0NAD FLAGELLATES is Locke-Egg-Blood m e d i u m i n its original form or m o d i f i e d i n various ways. Ringer solution or Intestinal trichomonad flagellates of ver0.7$ sodium chloride m a y be substituted for tebrate hosts m a y be cultured o n various m e d i a Locke solution, to cover the egg slant, and i n used for intestinal flagellates and amoebae. it 0.5 ed. 10, p. 260.) F o r Flemming, 16 to 20 minutes; sublimate acetic, 4 minutes; Champy, 25 minutes is the recommended time. Chen (1944b) after weak Flemming hydrolized 5 minutes at room temperature, then 15 minutes at 60°C. Rinse in cold HC1 solution. Rinse i n distilled water. S t a i n in fuchsin-sulfurous acid, 1 to 3 hours. (Prepare stain as follows: Bring 200 cc. distilled water to boil. Add 1 gram powdered basic fuchsin, stir for solution. Cool to 50°C. and filter. A d d 20 cc. of normal HC1 solution. Cool to 25°C., and add-1 gram potassium m e t a b i sulfite or anhydrous sodium bisulfite. Allow to stand in dark for 12 to 24 hours or more before using, w h e n it should have become yellow. If it is red, instead of yellow, do not use. K e e p well stoppered in dark.) W a s h i n 3 changes of dilute sulfurous acid (distilled water 200 cc.; 10$ solution of potassium metabisulfite or anhydrous sodium bisulfite, 10 cc.; normal HC1, 10 cc. 1 to 5 minutes each.) W a s h in distilled water. Pass through grades of alcohols to 95$Counterstain if desired in light g r e e n o r fast g r e e n FCF saturated in 95$ alcohol, 1 / 2 to 2 min. Complete the dehydration, clear, and mount. (See also De Tomasi, 1936; Rafalko, 1946.) F I X A T I O N FLUIDS. BENOIT FLUID aqueous uranium nitrate 5$ aqueous potassium dichromate Saturated mercuric chloride in 2$ osmic acid saline W a s h well in running water.

4 6 5 5'

parts parts parts parts

BOUIN F L U I D (PICRO-FORMOL-ACETIC) Saturated aqueous picric acid 75 cc. Formalin 25 cc. Acetic acid 5 cc. W a s h i n 50$, then 70$ alcohol until most of the picric acid is removed. BRASIL-DUBOSCQ MODIFICATION OF B O U I N F L U I D 80$ alcohol 150 cc. Formalin 60 cc. Glacial acetic acid 15 cc. Picric acid 1 gram This is m o r e penetrating for cysts, etc., than Bouin. It m a y be used hot.

The test is for thymonucleic acid, a constituent of chromatin. B y hydrolysis, certain constituents are broken down into aldehydes, and to these the Schiff reaction (fuchsin-sulfurous acid) is applied. The result is a red or violet stain, w h i c h is largely specific to chromatin, but not all chromatin gives the reaction and CARNOY F L U I D some other substances do stain. A contrast Glacial acetic acid 1 part stain is often used for nucleoli and cytoplasm. Absolute alcohol 6 parts Fix i n Flemming fluid, Champy fluid, subliChloroform 3 parts mate acetic, 6$ sublimate w i t h 2$ glacial acetic, W a s h out in 90$ alcohol. Penetration e x or other fixative. W e a k Flemming is good. cellent, destructive to cytoplasmic structures.

MATERIALS A N D METHODS IN THE STUDY OP PROTOZOA CHAMPY FLUID potassium dichromate chromic acid osmic acid

7 parts 7 parts 4 parts

Films m a y be fixed for 10 minutes or m o r e , "bulk material and tissues for several hours or m o r e . After Champy fluid or other chromeosmium or osmium fixative thorough washing is important. Films m a y be washed in running water for a n hour; if fixation has been prolonged, several hours m a y be better. Preparations m a y be improved if, after fixation and brief washing, they are placed f o r three days in 3$ aqueous potassium dichromate. After this, they should be washed i n running water for several hours, then bleached before staining. If the preparations are to be stained in iron haematoxylin (Regaud or Heidenhain), they should first be brought to 70$ alcohol and preferably left for at least several hours. DA FANO FIXATIVE Modified as used preliminary to silver nitrate impregnation b y Chatton and Lwoff, 1930. Freshwater forms Cobalt nitrate 1 gram Formalin 10 cc. Sodium chloride 1 gram Distilled water 90 cc. W a s h out in water. Marine forms Cobalt nitrate 1 gram Formalin 10 cc. S e a water 90 cc.

40

One part formalin and four parts 3$ aqueous potassium dichromate. One part formalin and three parts saturated aqueous mercuric chloride. Three parts 10$ formalin and one part 10$ nitric acid. After fixation, wash well in water. GELEI SUBLIMATE-DICHROMATE-ALUM F I X A T I V E Mercuric chloride 7 grams Potassium dichromate 1 - 2.5 grams Potassium alum 1 gram Distilled water 100 cc. W a s h out i n water used for fixation prior to silver impregnation methods (Gelei, 1939). HEIDENHAIN SUSA F I X A T I V E Stock solution: Mercuric chloride 4.5 grams Sodium chloride 0.5 grams Water 80 cc. To m a k e u p for use: S t o c k solution 80 cc. Glacial acetic 4 cc. Formalin 20 cc. Trichloracetic acid 2 grams W a s h out in 90$ alcohol. There is not m u c h sublimate precipitate; if present, it can be rem o v e d b y iodine in the alcohol. HERMANN F L U I D 2$ osmic acid 4 cc. 1$ platinum chloride 15 cc. Glacial acetic acid 1 cc. Acetic acid m a y be omitted if desired. W a s h In water.

HIRSCHLER F L U I D FLEMMING FLUID 2$ osmic acid 10 cc. Strong fixative. 1$ chromic acid 5 cc. 1$ chromic acid 15 cc. 3$ potassium dichromate 5 cc. 2$ osmic acid 4 cc. Ingredients as in Champy fluid, but the Glacial acetic acid 1 cc. proportions are different. W a s h well i n water. W e a k fixative 1$ chromic acid 25 cc. H O L L A N D E CUPRIC PICRO-FORMOL 1$ glacial acetic 10 cc. Distilled w a t e r 100 cc. 2$ osmic acid 5 cc. Copper acetate, neutral 2.5 grams Water 60 cc. Picric acid 4 grams Normal salt solution is sometimes u s e d for Formalin 10 cc. the fluids for endozoic protozoa. Glacial acetic acid 1.5 cc. 3. Flemming-without-acetic. The copper acetate is dissolved cold In W h e n chondriosomes and other lipoid elements w a t e r in a mortar. are to be preserved acetic-acid is omitted The picric acid is now added little by from the above formulas. little. A f t e r Flemming fixation, wash well in runThe formalin and acetic acid m a y be a d d e d ning water, as after Champy. shortly before use. The latter m a y be omitted if desired. FORMALIN W a s h out in water or 50$ alcohol. 10$ formalin can be used alone as a fixative. 0.75$ sodium chloride m a y be added if desired. A fixative for protozoa m a y be made u p of OSMIC A C I D 10 parts of 1 or 2$ osmic acid and 1 part formalin. Osmic acid is an excellent reagent to preFormalin m a y be used with other substances serve the f o r m of the body in m a n y protozoa, to as follows, m i x e d just before use: fix flagella, cilia, and cytoplasmic structures.

MATERIALS A N D METHODS IN THE STUDY OP PROTOZOA It causes a minimum of shrinkage and alters the structure of the cytoplasm less than do many, other fixatives. Killing and fixation m a y be accomplished b y exposure to osmic vapor, or by addition of 1 - 2$ aqueous solution. Osmic acid is an ingredient of m a n y fixation fluids, including Flemming, Champy, Hermann, and Hirschler. 10$ of formalin m a y be added to it. Osmic vapor fixation m a y be carried out by using a low glass vial bottom or a glass ring, cemented o n a slide, the diameter of the ring being a little less than a circular cover glass. The vessel is filled to within half a centimeter of the rim with 1 to 2j( osmic acid. The preparation o n the cover glass is placed over the vessel, which m a y be sealed by placing vaseline o n the rim. Exposure for flagellates ig from 10 seconds or less to 20 seconds; larger forms m a y be left 1 to 5 minutes or more. The preparation m a y be fixed subsequently in Flemming solution or a sublimate fixative; this separate fixation is often omitted. After fixation in osmic acid solution or vapor, the preparations should be w a s h e d 10 to 30 minutes in running water. PERENYI FLUID 95$ alcohol 3 parts 10$ nitric acid 4 parts 0.5$ chromic acid 3 parts According to Wenrich (l94l) this fixative m a y give good results for larger ciliates w h e n they are distorted by Schaudinn fluid. Petrunkevitch fluid is also recommended for these protozoa. PETRUNKEVITCH FLUID Saturated aqueous mercuric chloride Absolute alcohol Acetic acid Nitric acid

300 200 90 10

cc. cc. cc. cc.

SCHAUDINN FLUID Saturated mercuric chloride in water 2 parts 95$ or 100$ alcohol 1 part A small amount of glacial acetic acid (usually 2 to 5 per c e n t ) i s generally added. The stock should be kept without acetic acid, w h i c h can be added before use. Fixation m a y be at room temperature or at temperatures u p to 40° to 50° C. It has not b e e n found that there is any significant difference in fixation of m o s t protozoa b e t w e e n the results obtained at room temperature and higher temperatures. Schaudinn fluid m a y be diluted w i t h water to one-half or one-quarter strength, and from 2$ to 5$ acetic acid then added; the dilute fixative is very satisfactory for m a n y protozoa. After fixation, the sublimate should be rem o v e d b y placing the preparations in iodine in 70$ alcohol. SUBLIMATE ACETIC Saturated aqueous mercuric chloride Glacial acetic acid

9 5 cc. 5 cc.

Sublimate-alcohol-acetic is made with saturated mercuric chloride in 95$ alcohol. After fixation, the mercuric chloride is removed b y iodine in weak solution in alcohol, or by Lugol solution. WORCESTER FLUID 10$ formalin saturated with mercuric chloride Glacial acetic acid

9 parts 1 part

YOCUM FLUID 2 grams Mercuric chloride 1 gram Picric acid 110 cc. 95$ alcohol 20 cc. Ether 20 cc. Glacial acetic acid 50 cc. Formalin This fixative, w h i c h includes so m a n y substances, had best be m i x e d from stock ingredients before use. ZENKER F L U I D Potassium dichromate 2.5 grams Mercuric chloride 5 grams Water 100 cc. Glacial acetic acid 5 cc. The acetic acid should not be added until immediately before use. It m a y be omitted. Tissues are fixed 10 to 24 hours. Film preparations need not be fixed so long. As i n any sublimate fixative, iodine-alcohol m a y be u s e d to remove the crystals. •Wash well in water. F I X A T I O N FLUID MIXTURES FROM STOCK SOLUTIONS. M a n y fixatives are unstable and should be prepared shortly before use. The components of these fixatives m a y , however, be kept separately in solution. A convenient w a y of having f r e s h fixatives is, therefore, to keep the components separately and mix the fixation fluids as needed. G r a y (1933) recommended this procedure, keeping stock solutions as follows. The list is m o d i fied according to requirements for fixatives commonly used in protozoology. 1. F o r m a l i n (40$ formaldehyde). 2. Glacial acetic acid. 3. Chloroform. 4. Ether. 5. Absolute alcohol. 6. 2$ chromic acid i n water. 7« 7 - 5 $ potassium dichromate i n water. 8. 2$ osmic acid in 0.013$ potassium permanganate in water. 9. Saturated picric acid in water. 10. 1$ picric acid in 95$ alcohol. 11. 2.5$ copper acetate and 4$ picric acid i n water, copper acetate dissolved first. 12. 1$ picric acid and 1$ mercuric chloride in 95$ alcohol. 13- Saturated mercuric chloride in water. 14. Mercuric chloride 4.5 grams, water 80 cc, sodium chloride 0.5 grams.

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA It h a s o f t e n b e e n s u p p o s e d that it is a d v a n t a g e o u s to fix f o r r.elatively long p e r i o d s of t i m e , as in p r e p a r a t i o n of b l o c k s of t i s s u e , o r to u s e w a r m o r h o t f i x a t i v e s . W e n r i c h f o u n d that i n f i x i n g the o r d i n a r y F i x a t i o n f l u i d s are m i x e d as f o l l o w s : i n t e s t i n a l p r o t o z o a e x p o s u r e to the c o m m o n f i x a B o u i n : S t o c k 1, 25 co.; S t o c k 2, 5 oc.; S t o c k tives (as S c h a u d i n n f l u i d or B o u i n fluid) f o r 9, 7 5 cc. one m i n u t e w a s as s a t i s f a c t o r y as a l o n g e r F o r B o u i n - U r e a of M c C l u n g , a d d 1 g r a m u r e a . B r a s i l - D u b o s c q : S t o c k 1, 26 cc.; S t o c k 2, 6.5cc.; p e r i o d , a n d t h e r e w a s no a d v a n t a g e i n r a i s i n g S t o c k 10, 45 cc.; S t o c k 5, 15 cc; w a t e r 8 cc. the t e m p e r a t u r e a b o v e o r d i n a r y r o o m t e m p e r a t u r e . F i x a t i o n i n S c h a u d i n n f l u i d or B o u i n f l u i d d i C a r n o y : S t o c k 2, 10 cc.; S t o c k 3, 30 cc.; S t o c k l u t e d to o n e - h a l f s t r e n g t h g a v e f u l l y s a t i s f a c 5, 60 cc. tory results. Saturated mercuric chloride diC a r n o y - L e b r u n : S t o c k 2, 30 cc.; S t o c k 3, 30 cc.; l u t e d to one-half s t r e n g t h o r less g a v e b e t t e r r e S t o c k 5> 30 cc.; add 7 grams m e r c u r i c c h l o sults t h a n the full s t r e n g t h s o l u t i o n . ride. H o l l a n d e (1942) f o u n d that f o r small f r e e C h a m p y : S t o c k 6, 20 cc.; S t o c k 7 , l6 cc.; S t o c k l i v i n g f l a g e l l a t e s the b e s t p r e p a r a t i o n s w e r e 8, 22 cc.; w a t e r 33 cc. o b t a i n e d b y f i x i n g l e s s t h a n a m i n u t e (10 to 20 E r l i c k i : S t o c k 7 , 33 cc.; w a t e r , 67 cc.; 1 g r a m s e c o n d s i n c o l d S c h a u d i n n f o r C y a t h o m o n a s ; 1 to copper sulfate. 5 s e c o n d s f o r C e r c o b o d o in a l c o h o l i c S c h a u d i n n Flemming: and Benoit fluid). S t r o n g s o l u t i o n : S t o c k 2, 5 cc.; S t o c k 6, It m a y b e s u p p o s e d ( t h o u g h trial w i t h the 3 8 cc.; S t o c k 8, 20 cc.; w a t e r 3 8 cc. s p e c i f i c m a t e r i a l to b e u s e d is n e c e s s a r y ) that W e a k s o l u t i o n : S t o c k 2, 0.1 cc.; S t o c k 6, f o r f i x a t i o n of small p r o t o z o a that are f u l l y 1 2 . 5 cc.; S t o c k 8 , 5 cc.; w a t e r 83 cc. e x p o s e d to the a c t i o n of t h e r e a g e n t t h e r e is n o W i t h o u t a c e t i c : S t o c k 6, 40 cc.; S t o c k 8, a d v a n t a g e i n h e a t i n g o r i n p r o l o n g i n g the time 21 cc.; w a t e r 40 cc. G e l e i : S t o c k 7, 25 cc.; w a t e r 7 5 cc.; a d d 1 g r a m b e y o n d a few m i n u t e s at m o s t . A d i l u t e d f i x a t i v e m a y g i v e as g o o d o r b e t t e r r e s u l t s t h a n t h e f u l l p o t a s s i u m alum, 7 g r a m s m e r c u r i c chloride. strength solution. Gils.on: S t o c k 1 6 , 1.5 cc.; S t o c k 2, 0.4 cc.; F o r f i x a t i o n of f r e e - l i v i n g a n d e n d o z o i c S t o c k 13, 35 cc.; w a t e r 50 cc.; S t o c k 5, 7 cc. c i l i a t es (Paramecium, opalinids) in preparation H e i d e n h a i n S u s a : S t o c k 14, 80 cc.; S t o c k 2, f o r s t a i n i n g of n u c l e i a n d c h r o m o s o m e s C h e n 4 cc.; S t o c k 1, 20 cc.; add 2 g r a m s t r i c h l o (l9^^a) r e c o m m e n d e d f i x i n g i n S c h a u d i n n f l u i d r a c e t i c acid. In H e r m a n n : S t o c k 2, 5 cc.; S t o c k 14, 38 cc.; S t o c k h e a t e d to 4 0 ° to 5 0 ° C. f o r 5 to 15 m i n u t e s . fixing Plasmodium in blood films from birds Chen 8 , 2 cc.; w a t e r 38 cc. (1944b) u s e d m o d i f i e d w e a k F l e m m i n g f l u i d at H i r s c h l e r : S t o c k 8, 50 cc.; S t o c k 7 d i l u t e d w i t h a n equal a m o u n t of w a t e r , 25 cc.; S t o c k 6 d i - r o o m t e m p e r a t u r e f o r 1 to 4 h o u r s . l u t e d w i t h a n equal amount of w a t e r , 25 cc. H o l l a n d e : S t o c k 11, 90 cc.; S t o c k 1, 10 cc.; FLAGELLATA. S t o c k 2, 1 . 5 cc. 15- 2$ p l a t i n u m c h l o r i d e , s t o r e d i n a m b e r bottle. l6. C o n c e n t r a t e d n i t r i c a c i d 4 p a r t s , w a t e r 1 part.

K o l a t c h e v : S t o c k 6, 20 cc.; S t o c k 7 , l8 cc.; S t o c k 8, 20 cc.; w a t e r 42 cc. P e t r u n k e v i t c h : S t o c k l6, 2 cc.; S t o c k 2, 17 cc.; S t o c k 5j 3 8 cc.; w a t e r , 50 cc.; a d d 7 g r a m s mercuric chloride. S c h a u d i n n : S t o c k 13, 67 cc.; S t o c k 5, 33 cc.; S t o c k 2, 2 cc. to 5 cc. a d d e d if d e s i r e d . Y o c u m : S t o c k 1, 5 cc.; S t o c k 2, 20 cc.; S t o c k 12, 55 cc.; S t o c k 4, 20 cc. Yocum, Gray's Modifications: F o r P r o t o z o a , S t o c k 12, 10 cc.; S t o c k 1, 5 cc.; S t o c k 4, 3 cc.; S t o c k 2 , 2 cc. F o r d e l i c a t e l a r v a e , S t o c k 12, 10 cc.; S t o c k 1, 5 cc.; S t o c k 4, 2 cc.; S t o c k 2, 1 cc. F o r f o r m s w i t h t h i c k c u t i c l e , S t o c k 12, 10 cc.; S t o c k "1, 5 cc.; S t o c k 4, 1 cc.; S t o c k 2, 4 cc. Z e n k e r : S t o c k 2 , 5 cc.; S t o c k 7 , 30 cc.; S t o c k 13, 70 cc. FIXATION TIME AND

TEMPERATURE.

A n i m p o r t a n t p a p e r o n t h e role of t h e s e f a c t o r s i n f i x a t i o n of p r o t o z o a is that b y W e n r i c h (1941).

The following procedures were recommended b y H o l l a n d e (19^2) f o r the s t u d y of f r e e - l i v i n g flagellates. M'ake c o v e r g l a s s p r e p a r a t i o n s b y f l o a t i n g covers o n the s u r f a c e or i m m e r s i n g i n c u l t u r e . Place t h e c o v e r i n f i x a t i v e i n a P e t r i dish. T h e t i m e of f i x a t i o n s h o u l d b e v e r y s h o r t (in s e c o n d s r a t h e r t h a n m i n u t e s ) to g i v e the c l e a r est p r e p a r a t i o n s . Fix in Benoit, Schaudinn with acetic, or other fixative. W a s h i n r u n n i n g w a t e r 15 m i n u t e s . Stain in iron haematoxylin or other stain. F o r the c h o n d r i o s o m e s , place a d r o p w i t h the f l a g e l l a t e s o n the s l i d e , a d d a d r o p of H e r m a n n f i x a t i v e , m i x , s p r e a d out, a n d d r y ; w a s h rapidly in running water; stain in Altmann fuchsin. F o r s e c t i o n s , f i x i n b u l k 2 to 10 m i n . i n Hirschler fixative, weak Flemming, Benoit or other reagent. Place i n the f i x a t i v e i n a small d i s h , i n w h i c h f l a g e l l a t e s c o l l e c t at t h e b o t t o m b y s e d i m e n t a t i o n ; b y m e a n s of a p i p e t t e , d r a w off f l u i d a n d r e p l a c e at i n t e r v a l s b y w a t e r a n d a series of a l c o h o l s , 5 m i n . each. Clear in toluol. D r a w off t o l u o l c a r e f u l l y , c o l l e c t the

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA flagellates on a small spatula and place in paraffin In tin or other capsules at 600 C. Impregnate 5 to 15 min. in capsule. Cut sections at 10 to 15 Ms rarely 5f- Stain in iron haematoxylin, counterstain eosin or other contrast stain. After osmic fixatives, preparations may fail to stain. To overcome this difficulty, after dissolving paraffin from the sections, carry them to water, place in 0.25$ potassium permanganate 12 hours, rinse in distilled water, place in solution of equal parts of 0.5$ oxalic acid and 0.5$ potassium sulfite until the brown color disappears. Osmic vapor fixation was found good for Bodo and endozoic flagellates, but not for most free-living flagellates. Fixation in vapor of iodine crystals in 95$ alcohol at 30° C. was good for Tetramitus, but not for general use. For nuclear structure and mitosis, MANN METHYL BLUE EOSIN STAIN gave good results. Preparations of intestinal flagellates are made ordinarily by fixation in Schaudinn's fluid, or other fixative and staining by Heidenhain's iron haematoxylin. Modified haematoxylin procedures give good results with particular forms. Preparations which may reveal certain structural details not otherwise demonstrated, and show flagella with unmistakable clarity, can often be obtained by use of Bodlan's activated protein silver. A satisfactory type of protargol must, however, be used. Discussion of some matters applicable also to other flagellates is given under TRICHOMONAD FLAGELLATES and TERMITE FLAGELLATES. The dry and wet methods discussed under GIEMSA STAINING may be used for blood and tissue flagellates, and are useful also for special purposes in study of some intestinal flagellates. F ORAMINIFERA. For picking up the specimens a fine, moistened brush may be used, one in which the bristles can be drawn to a fine point; red sable brushes, size 00 are recommended. Slides for mounting Foraminifera, with openings in pasteboard over a dark background, can be purchased from supply houses or made. Slides with a rectangular opening over a black background marked off in white in numbered sections are convenient. A coating of gum tragacanth dissolved in warm water can be spread on the mounting surface and dried. When the specimen is transferred to the mounting surface by a moist brush, the gum is softened by the moisture and specimen Will become attached. The pasteboard mounts may be covered by glass slides and the edges fastened by strips of gummed cloth or paper. Metal holders are made into which the slides can be slipped; with these it is possible to remove the slides to remount or add to the specimens if desired. In making preparations for cytological

study, the test must be decalcified. The specimens are fixed in a suitable reagent. Decalcification is accomplished by acids in the fixative, or by subsequent treatment in acid alcohol, depending on the species that is being studied. For Spirillina, Myers (1936) used modified Schaudinn fluid with 25$ acetic acid added immediately before use, heated to 65° C. and allowed to act for 2 to 5 minutes. Tretomphalus was fixed by Myers (19^3) in Schaudinn fluid without acetic, and decalcified after fixation by hydrochloric acid in 85$ alcohol, an amount of HC1 sufficient to dissolve the test without causing noticeable effervescence. Total mounts may be stained in alum haematoxylin (as Ehrlich acid haematoxylin), or in Heidenhain iron haematoxylin. If sections are desired, the decalcified specimens may be imbedded. Myers infiltrated Tretomphalus in celloidin, imbedded it in that substance, hardened the celloidin in 95$ alcohol, placed the mass in acidified 85$ alcohol to complete decalcification, clearing in carbol-xylol, then reimbedded the mass in paraffin. FORMALIN. F o m a l i n or formol is a 37$ to 40$ solution of formaldehyde gas in water. It is used as a preservative for macroscopic specimens and as a fixative. As a preservative, formalin is diluted to 4$ or 5$ of the commercial solution. Specimens are hardened and cannot be restored to shape if deformed at this concentration. The vapor of formalin is irritating, and causes severe reactions in some persons. The odor will be suppressed if specimens are washed and placed in water to which a little ammonia has been added. Specimens become more pliable and the irritating effect of the preservative is reduced by soaking in 3$ carbolic acid. As a fixative, formalin is generally used in a dilution of 10$ of the commercial liquid. Ten per cent formalin then means about 4$ formaldehyde. Concentrations of 20$ and 25$ formalin are sometimes used. It gives a general fixation, is adopted for certain special uses, but for cytological work is generally combined with other fixation reagents. Formalin may contain some formic acid, which is undesirable for some purposes. Neutral formalin may be obtained; the liquid can be kept neutral by keeping calcium carbonate, tied in a muslin bag, at the bottom of the jar. GIEMSA STAINING. Giemsa stain can be bought already made up, or a stock solution can be mixed as follows: Azur II - eosin 3 grams Azur II 0.8 grams Glycerine 125 or 250 cc. Methyl alcohol 375 or 250 cc.

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA The amounts of glycerine and methyl alcohol vary in different formulas. The dye may be dissolved in glycerine at 60°C. and warmed alcohol added. The pH of the staining solution, in Giemsa and other Romanowsky stains is important. Solutions at different pH may stain different structures in comparable material. pH may be adjusted by using buffer solutions for dilution of the stock staining solution. Giemsa may be used in staining of dried films or of wet-fixed films. STAINING DRY BLOOD FILMS BLOOD FILMS may be prepared thin or thick, as described under that heading. Thin films Thin films may be fixed l/2 to 3 minutes by covering them with methyl alcohol; or they may be fixed in May-Grünwald solution, which is excellent as a preliminary to staining. (MayGrünwald solution is a saturated solution of methylene blue eosinate in methyl alcohol; it may be made up, if the eosinate is not available, by mixing equal parts of 1.25$ aqueous eosin solution and aqueous methylene blue, leaving 24 hours, filtering, drying the precipitate on the filter paper and dissolving it in 200 cc. of methyl alcohol). The fixing solution should not be allowed to dry, and the time should not exceed that recommended. Drain off the remaining methyl alcohol, or dilute May-Grünwald by adding an equal amount of neutral distilled water and leaving 1 minute before draining off. Prepare the diluted stain fresh by adding 1 drop of the stock solution to 1 cubic centimeter of water at suitable pH, or 3 drops to 2 cc. The stain should not be more concentrated. After adding the stain, the fluid should not be stirred or agitated excessively. Buffered distilled water at the desired pH may be used for stain dilution. The stain may be placed on the film with a pipette, or the slide or cover glass may be placed in a dish of stain. If placed film side facing downward, formation of a precipitate on the preparation will be avoided. Allow the stain to act for 30 to 45 minutes, or more or less according to the results desired. Wash off the stain from the slide with a jet of water from a pipette or by dipping the slide Into water. Any precipitate must be removed from the slide. If the preparations are overstained, they can be differentiated by prolonged washing in water, by boric acid, or by 1% 30dium phosphate, dibasic. The vigor of action increases in the order given. Dry the slides by draining and in air. The films may be kept dry, or covered by neutral balsam or other mounting medium and a #0 or #1 cover glass.

44

Thick films Thick films may be stained by Giemsa solution in the same manner as thin films, but without previous fixation in alcohol. For the staining solution Wilcox (1943) recommended 50 parts of buffered distilled water (pH 7.0 to 7.2) to 1 part of Giemsa stock solution. The slides are set on end immersed in the solution and stained 45 minutes. They are then placed in neutral distilled water for 3 to 5 minutes. After drying and before immersing in the stain, thick films may be dehaemoglobinized by placing them in a fluid consisting of 4 parts of 2.5$ acetic acid and 1 part of 2$ crystalline tartaric acid, until the color of the film is grayish white. The fluid is then drained off and the preparation fixed 3 or 4 minutes with methyl alcohol. The alcohol is drained off, the film washed thoroughly in distilled water, and stained with Giemsa (l drop to 1 cc.) 15 minutes or longer. The preparation is differentiated, if necessary, in distilled water and dried in air, without blotting. STAINING WET-FIXED PREPARATIONS Giemsa stain may be used as a nuclear and cytoplasmic stain for properly fixed material intended for cytological study. Fixation may be in Schaudinn fluid, sublimate, or Zenker fluid, all preferably without acetic acid. After fixation, the sublimate is washed out with iodine alcohol, the preparation rinsed, and then placed for 10 minutes or so in 0.5$ aqueous sodium hyposulfite. The slides should then be washed well in water. Brasil-Duboscq fluid may also be used for fixation; after this iodine is not necessary, but the picric acid should be well washed out. For staining, place the preparations in a solution made from Giemsa and neutral distilled water or buffered water at pH 7-0 to 7-2 in the proportion of 1 drop stain to 1 cc. water. Stain for 10 to 45 minutes or longer, one or more hours, according to the result desired. The staining solution may be replaced by a fresh solution after a half hour or so. Wash the preparations rapidly in distilled water. Differentiate if necessary in 1$ boric acid or 1$ sodium phosphate, dibasic. Pass through mixtures of acetone and xylol in the proportions of 95 acetone - 5 xylol, 70-30, 50-50, and pure xylol. Following osmic or chromic acid fixation, the preparations should be placed in 1 to 2% aqueous ammonium molybdate for 1 to 2 mln., washed in distilled water 2 to 5 min., then stained as above. Gelei's modifications recommend differentiation in 96$ alcohol. GLYCERINE JELLY. 40 grams gelatin soaked in 200 cc. distilled water 2 hours. 250 cc. glycerine added,

MATERIALS A N D METHODS IN THE STUDY OP PROTOZOA stirred, heated 10 to 15 minutes. 5 grams of carbolic acid m a y be added as a preservative. GLYCHROGEL. M a y be used in place of glycerine jelly in mounting preparations from aqueous media. Dissolve 0.2 grams chrome alum in 30 cc. distilled water. Dissolve 3 grams granulated gelatin in 50 cc. hot water, and add to the alum solution. A d d 20 cc. glycerine, w a r m and mix, then add a crystal of camphor. W a r m for use.

GLYCOGEN. Iodine will stain glycogen and "paraglycoThe gen" mahogany brown or reddish brown. m e t h o d is not specific for glycogen, as amyloids and other substances will stain similarly. A control can be set u p b y allowing saliva to act o n the preparation at 37° for 15 to 30 minutes, changing the saliva several times. W a s h thoroughly in water. The enzyme removes the g l y c o gen, so that the iodine reaction is subsequently not obtained. For the iodine reaction L U G 0 L SOLUTION is used. It m a y be added to the fresh preparation; or the material m a y first be fixed. F i x a t i o n of preparations for glycogen staining can be done in Carnoy fluid (3 parts absolute alcohol and 1 part glacial acetic) or better in a fluid consisting of 9 parts of absolute alcohol (pure or saturated with picric acid) and 1 part of neutral formalin. BEST'S CARMINE is useful to stain glycogen i n fixed preparations. It will also stain other substances, and control b y salivary action is necessary for identification of a substance as glycogen. The fixed preparations are stained in Ehrlich or Delafield haematoxylin and differentiated in acid alcohol. T h e y are then put in-diluted Best's Carmine (2 parts stock solution, 3 parts concentrated ammonia, 6 parts of methyl alcohol) for 5 minutes or more. Glycogen is stained bright red. If necessary, the preparations can be differentiated after staining in a fluid consisting of 4 parts of absolute ethyl alcohol, 2 parts of methyl alcohol, and 5 parts of water. The differentiating fluid is allowed to act for several minutes or until no more red stain comes out. The preparations are then placed in 80$ alcohol, dehydrated, cleared and mounted.

F e u l g e n technique and are w a s h e d in running water. They m a y be dehydrated, cleared and mounted; or m a y be stained first in alum-haematoxylin (Ehrlich or Delafield) or other nuclear stain. Glycogen is stained reddish violet; starch, cellulose and other substances m a y also be stained. (See Bensley, 1939»)

G0LGI BODIES. Various substances in protozoa have b e e n identified as Golgi bodies following their demonstration by methods used for the m e t a z o a n Golgi apparatus. The m o s t reliable of these methods employ osmic acid. Fixation m a y be in a mixture of equal parts of saturated mercuric chloride and 1$ or 2$ osmic acid; or in a n osmic-dichromate fixative consisting of 2 parts 3$ potassium dichromate, 2 parts 1$ chromic acid, 1 part 2$ osmic. Fixation for 1 to 3 hours should be sufficient for protozoa of ordinary size w h i c h are not in tissue. A f t e r thoroughly washing out the fixative i n water, specimens are placed in 2$ osmic acid and kept at a temperature of 25° to 3 5 ° C. g e n erally for one or two weeks. The H i r s c h l e r technique calls for 12 to 16 days at 25°C.; the Ludford technique employs a temperature of 35°> w i t h 1 day in 2$ osmic, 1 day in 1$, and the third d a y in 0.5$; the Kolatchev m e t h o d requires 8 hours in 2$ osmic at 40°, followed b y 3 to 5 days at 35°C. The methods used f o r tissue m a y be modified according to the requirements f o u n d necessary f o r the protozoan material being studied. GRAM SOLUTION.

(See IODINE.)

GREGARINES. All stages of monocystids m a y be f o u n d in seminal vesicles of earthworms at the proper season, generally i n the spring. A t other times there m a y be only cysts. Occurrence of g r e g a rines i n other hosts m a y be seasonal. W h e n the hosts are confined in laboratory cultures, the incidence and amount of infection m a y be increased.

Monocystids m a y be studied b y dissecting out seminal vesicles of earthworms in 0.6$ sodiu m chloride or Ringer solution and examining in f r e s h or prepared smears or b y preparing sections of the organs. Excellent results in staining g l y c o g e n are g i v e n b y Bauer's method, w h i c h also, however, is The forms i n the intestine of their host not specific. Fixation is in B o u i n fluid to m a y be studied in f r e s h preparations after exw h i c h 1.5 grams of chromic acid have b e e n added tracting and opening the gut, or in f i x e d and to 100 cc., or in a mixture of formalin and abso- stained films. F i x a t i o n of the unencysted forms lute alcohol pure or saturated with picric acid, m a y be done i n Brasil's alcoholic B o u i n fluid or 1:9 as above. The fixed preparations are placed other reagent. F o r nuclei, stain in carmine or in chromic acid for an hour, washed in runalum haematoxylin. ning water, then placed for 10 to 15 minutes in Cysts m a y be f o u n d i n the fecal deposits of f u c h s i n sulfurous acid as used in the F E U L G E N the infected hosts. Cysts m a y be picked out N U C L E A L REACTION. The slides are then placed in w i t h a fine pipette after immersing feces in 3 changes of dilute sulfurous acid as in the water, and removing to a small glass dish. They

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA m a y be kept dry in a watch glass placed in a m o i s t chamber floored b y moist towel paper or filter paper. A piece of black paper under the w a t c h glass will aid visibility of the cysts, w h e n they are examined with a stereoscopic microscope. Preparations of the cysts can be made by crushing them o n cover glasses or slides, preparing films, and fixing in Schaudinn fluid, Brasil-Duboscq, Zenker fluid or other fixative. Subsequent staining is b y Heidenhain's iron haematoxylin, the Feulgen nucleal reaction, o r other stain. These film preparations are best f o r studying detail of nuclear changes. Cysts m a y be fixed whole and sectioned. F o r good penetration, Brasil-Duboscq m o d i f i c a tion of B o u i n fluid gives superior results. A f t e r fixation, the cysts are washed and carried to 70$ alcohol, then tertiary butyl alcohol or d i o x a n is slowly added. After the proportion of butyl alcohol or dioxan has become high, they m a y be placed in pure solution of one of these reagents, and several changes made. Paraffin is subsequently added slowly, in a n Incubator, until the change to pure paraffin is made. To aid penetration of paraffin, it m a y be desirable to slightly cut or puncture the fixed cysts after they have b e e n hardened in alcohol. Sections are stained in Heidenhain's iron haematoxylin. Early stages of encystation m a y be studied b y selecting gamonts w h i c h are associated in pairs and show rotating movements, placing them i n f r e s h egg albumin in a depression slide, and covering w i t h a cover glass.

HAEMATOXYLIN STAINING IRON HAEMATOXYLIN STAINING Heidenhain M e t h o d F o r general purposes, iron haematoxylin is the most important stain for preparation of protozoa. It can be used after any m e t h o d of fixation, but in some instances different results are obtained after certain fixatives. Schaudinn fluid has b e e n widely used in protozoology; after it, iron haematoxylin m a y give a clear differentiation of various structural elements, but the fixative is destructive to other elements. After Heidenhain Susa fixative it m a y also give sharp differentiation. Flemm i n g fixative is valuable for m a n y purposes; and Hollande fixative has g i v e n excellent results i n flagellates. The fixation to be used, b y these and other fluids, must be adapted to the material and to the structures it is desired to bring out. Important variations of method i n iron haematoxylin staining are: 1. Use of aqueous solutions of iron alum and haematoxylin. This is the usual method. The mordant is 3$ to 5$ solution of iron alum (ferric ammonium sulfate). The crystals are pale violet in color. Where the surface is oxi-

46

dized, it is yellow. This yellow part should n o t be used. Do not use heat for solution. Iron alum is best if freshly prepared. Solutions cannot be kept v e r y long. The stain is a n 0.5$ or 1.0$ solution of haematoxylin. This is not good for staining w h e n freshly prepared, but m u s t be "ripened". The haematoxylin crystals should be from a lot that is k n o w n to be good for staining. The solution should stand at least for 3 to 6 weeks. A stock solution m a y be prepared b y dissolving 10$ of crystals in 95$ or 100$ alcohol. It will r i p e n after several months and m a y be good f o r m o r e than a year, but it will not keep indefinitely. Overoxidation m a y be prevented, after ripening is sufficient, by adding a very small amount of sodium bisulfite. The stock is diluted w i t h distilled water, after w h i c h it should stand for a time (perhaps 2 weeks) before being u s e d for staining. F r e s h l y prepared solutions m a y , however, be ripened quickly b y adding oxidizing agents; artificially oxidized solutions m a y deteriorate m o r e rapidly than those naturally oxidized, so stock to be kept should not be so treated. For rapid ripening 3 to 5 drops of 5$ aqueous potassium permanganate m a y be added to 100 cc. of the staining solution, or 5$ of a 1$ solution of potassium permanganate. Hydrogen peroxide m a y also be used, 5$ added to the staining solution. 2. Use of alcoholic solutions of iron alum and haematoxylin. The time of staining m a y be 10 minutes or longer. The same precautions m u s t be taken regarding ripening and quality of stain. 3. Use of a solution of haematein instead of haematoxylin. This is the substance w h i c h is formed in the process of ripening of haematoxylin, so a solution of h a e m a t e i n has m o r e precisely k n o w n staining properties. It cannot be k e p t , but m u s t be freshly prepared as needed. It m a y be used in 1$ solution in 70$ alcohol. See below. Adjustment of the pH of the staining solution. The staining solution m a y be m a d e alkaline b y addition of a "pinch" of sodium b i carbonate o r 3 drops of saturated lithium carbonate to 100 cc.; or f o r more precise control, the solution m a y be buffered. The color of the stain and the elements of the cell that are stained d i f f e r w i t h the pH. In iron haematoxyl i n staining of protozoa, where various cytoplasmic elements must be brought out, it is desirable to overstain in alkaline solutions and then differentiate. More precise control of pH of the staining solution is advantagedus for special purposes. 5. Differentiation can be carried out i n iron alum solution or in acids. Use of acids m a y obviate the difficulty of having a yellow o r b r o w n general coloration of the cytoplasm w h i c h m a y h a p p e n w h e n prolonged iron alum destaining

MATERIALS A N D METHODS IN THE STUDY OP PROTOZOA is necessary. Often, however, excellent differentiation is given b y iron alum. Saturated aqueous picric acid m a y be used. Hydrochloric or nitric acid, 0.2$ to 0.5$ is a good differentiating agent for protozoa. If it is necessary to reduce the time for differentiation, as for large ciliates, stronger solutions m a y be used. After the slide has been in acid, it should be rinsed and placed in a weak solution of ammonium hydroxide or 0.1$ sodium bicarbonate. If the acid is not neutralized it will damage the preparation. Iron alum must be thoroughly washed out in running water. 6. Various short-cut methods to reduce the time of staining have b e e n proposed. The methods that give the best results are usually the only ones that are w o r t h any time; and actually the short-cut methods are not appreciably time-saving, w h e n all things are considered. A laboratory schedule for iron haematoxylin staining m a y be carried out as follows: 1. W a s h out the fixative. Bring to w a t e r if in alcohol. 2. Place in 4$ aqueous iron alum. 10 minutes or more. 3. W a s h in distilled water. Place in 0.5$ aqueous haematoxylin f o r a period of time, at least equal to that of mordanting. Several hours to a d a y in the mordant and stain m a y be desirable. 5- W a s h in distilled water. 6. Differentiate in 2$ iron alum or i n 0.2$ to 0.5$ HC1. Put a slide on the m i c r o scope, add the reagent, and watch it. W h e n nuclei can be seen destaining is generally sufficient. 7- If acid has b e e n used, neutralize in 0.1$ sodium bicarbonate or weak ammonium hydroxide in water. 8. W a s h thoroughly in several changes of water, or in running water. 9. 30$ alcohol - 2 minutes or more. 10. 50$ alcohol - 2 minutes or more. 11. 70$ alcohol - 2 minutes or more. 12. 90$ alcohol - 2 minutes or more. 13. 100$ alcohol - 2 minutes or more. 14. F r e s h 100$ alcohol - 5 minutes. 15. Equal parts of absolute alcohol and xylol or toluol - 5 minutes. 16. Xylol or toluol - 5 minutes. 17« Mount in balsam or clarite. Use only # 0 or #1 cover glass. IRON HAEMATEIN STAINING De Freitas, 1936; m o d i f i e d Honigberg, 19^7. 1. F i x a t i o n in Schaudinn, Zenker, Helly, B o u i n fluid or other fixative. B o u i n fluid and Hollande fluid are good. 2. W a s h out fixative. 3. Mordant 1 hour i n 0.5$ iron alum i n 70$ a l cohol. (0.5 gram iron alum dissolved in 30 cc. distilled water, 70 cc. absolute a l cohol added.) This solution should be pre-

5.

6. 7.

8.

9«

47

pared not earlier than the day before use. Rinse in 70$ alcohol. Stain in the following solution, 1 hour: H a e m a t e i n (l$ in 70$ ethyl alcohol) 2 parts Buffer solution pH J.6 1 part (for general purposes; the pH m a y be adjusted according to the requirements of the material.) W a s h in 70$ alcohol. Differentiate in saturated picric acid in 95$ alcohol, or with 0.2$ to 0.5$ h y d r o chloric acid or nitric acid in 70$ alcohol; or pass through 50$ and 35$ alcohol to water and differentiate in saturated aqueous picric acid or 0 . 2 $ to 0 . 5 $ hydrochloric or nitric acid i n water. Place in two o r three changes of 70$ alcohol with 0.1$ sodium bicarbonate; or if second procedure has b e e n followed i n 7> neutralize i n water with a little ammonium hydroxide or 0.1$ sodium bicarbonate. Dehydrate, clear and mount. COUNTERSTAINING

Counterstaining after iron haematoxylin is o f t e n not advantageous in preparation of protozoa w h e n it is desired to observe m a x i m u m detail of cytological structure, since staining of the cytoplasm obscures the picture. Counterstaining properly controlled m a y , however, aid in demonstration of certain cytoplasmic elements, w h e n simultaneous demonstration is desired. Acid fuchsin can be used in 0.5$ solution i n water. The s t a i n can be m a d e more permanent in subsequent treatment b y placing the preparation afterwards in w a t e r with a little HC1. Overstain can be removed by washing in alkaline water, the process stopped i n acid water. Indulin saturated in 80$ alcohol, light g r e e n saturated in 95$ alcohol, 0.5$ aqueous congo red, or other plasma stain m a y be used. ALUM HAEMATOXYLIN STAINING Several solutions for haematoxylin staining are made up w i t h alum and haematoxylin combined. I n several of the formulas glycerin is added. I n Delafield's formula there is also methyl alcohol. Ripening can take place naturally, w h i c h will require at least several weeks; or, as i n several of the formulas, sodium or potassium iodate or hydrogen peroxide m a y be added to bring this about at once. Some of the stains are acidified b y addition of acetic o r some other acid; t h e y m a y be superior for nuclear staining. The stains m a y be diluted w i t h w a t e r to l/2 or l/3, or even as little as l/20 of t h e i r original strength. D i l u t i o n is especially desirable f o r Delafield haematoxylin. Overstaining is corrected b y destaining in 0.5$ HC1 (or more dilute solutions) in w a t e r o r

MATERIALS AND METHODS IN THE STUDY OF PROTOZOA 70$ alcohol. After use of acid, the preparations are placed until blue in alkaline tap water or in 0.1$ sodium bicarbonate. Delafield alum haematoxylin To saturated aqueous ammonia alum 400 cc. Add solution prepared b y dissolving 4 grams of haematoxylin in 25 cc. of 95$ alcohol. Expose to light and air 3 or 4 days, in a large beaker. Add 100 cc. glycerine and 100 cc. methyl alcohol. Allow the mixture to stand until it becomes dark. This m a y take several weeks. If rapid ripening is desired, add 10 cc. of hydrogen peroxide. Filter, and transfer to a well stoppered bottle. For staining, it is best to dilute the solution to l / 2 , 1 / 3 or less with distilled water. Ehrlich acid haematoxylin Dissolve 2 grams haematoxylin in 100 cc. absolute or 95$ ethyl alcohol. A d d 10 cc. glacial acetic acid; 100 cc. glycerine; then a solution of 3 grams ammonia alum or potassium alum in 100 cc. water. Expose to light and air until it becomes dark. This may take several weeks, after which it should be kept in a well stoppered bottle and will last for years. If rapid ripening is desired, 0.4 grams of sodium or potassium iodate m a y be added to the fresh solution. The same result m a y be achieved by using 0.4 gram haematein in making up the stain, instead of the two grams of haematoxylin. Mayer haemalum Distilled water 1000 cc. Haematoxylin 1 gram Sodium or potassium iodate 0.2 gram Potassium or ammonia alum 50 grams Dissolve at room temperature. For acid haemalum, add 1 gram citric acid and 50 grams chloral hydrate, or add up to 2$ acetic acid. Acid haemalum may keep better and give a more precise nuclear stain. TUNGSTIC HAEMATOXYLIN STAINING This gives progressive staining, and destaining is generally not necessary. It does not give the critical staining of cytoplasmic structures that can be obtained by iron haematoxylin. (See Dobell, 1942.) Mordant in 2$ phosphotungstic acid in distilled water, 10 minutes to several hours. Wash in several changes of distilled water. Stain in 0.2$ haematoxylin, 10 minutes to several hours. Wash, dehydrate, and mount.

HYDROGEN-ION CONCENTRATION, DETERMINATION OF In protozoology, hydrogen-ion concentration must be considered in adjustment of culture media for optimum growth, in staining, and in determination of the ecological conditions under which the organisms live. Determination of pH m a y be done by the colorimetric method or by an electrometric method. In the colorimetric method the solution to be determined is mixed with an indicator dye solution, and the color given Is compared with those of a series of mixtures of the same dye with buffer solutions of known pH. The buffer solution with the same color as the solution to be tested has the same pH value. Indicator dyes that m a y be used in the concentration given except in extraordinary pH ranges, are: Approximate pH range Concentration 1. Brom phenol blue 2.8 - 4.2 0.04$ 2. Brom cresol green 3.6 - 5.2 0.02$ 3. Chlor phenol red 4.8 - 6.4 0.04$ 4. Brom thymol blue 5-9 - 7.5 0.04$ 5. Phenol red 6.8 - 8.4 0.02$ 6. Cresol red 7.1 - 8 . 7 0.02$ 7 . Thymol blue 8.1 - 9-7 0.04$ (also 1.2 - 2.8) Dyes 3, 4, 5 and 6 are sufficient for most purposes. They m a y be made up in water or 95$ alcohol. When unbuffered or poorly buffered solutions are to be tested, accuracy requires that the indicator solutions be adjusted so as to have the color in the midpoint of their pH range. This may be done by adding N/20 sodium hydroxide (2 grams in 1000 cc. water), or dilute acid if that is necessary, until the color Is that at the midpoint of the range in the color standards for that indicator. A color chart giving the colors of indicators in solutions of known pH is given in Clark (1928). To prepare the color standards, buffer solutions of a selected range of known pH values are placed in 10 cc. amounts in consecutive tubes, and 0.5 cc. of the indicator solution suitable for each buffer is added. To test an unknown solution, an amount of 10 cc. is placed in a tube of the same kind as those that contain the color standard solutions and 0.5 cc. of an indicator is added, using an indicator in the range of that unknown, as determined by a preliminary test. For the preliminary test brom thymol blue may be used first, to show whether the unknown Is acid or alkaline. If its color comes at either end of its range, the indicator next to it at that end should be used; and so on until a color intermediate between the extremes of the Indicator range is obtained. To make color comparisons, two standard tubes of color close to the one of unknown pH

MATERIALS A N D METHODS IN THE STUDY OP PROTOZOA are placed o n either side of it, and the group held toward a source of light. A block comparator m a y be used in w h i c h vertical holes hold the tubes, and observation is made through a window and holes crossing the vertical holes at right angles so that precise comparison is possible. Color standards will not keep in open tubes. T h e y will last longer, but not indefinitely, if closed promptly w i t h rubber stoppers or sealed b y fusing the glass, and kept i n the dark when not in use. Electrometric methods of determining pH are carried out by special apparatus. The results g i v e n are more exact than those obtainable b y colorimetric methods. (See Clark, 1928; Cowdry, 19^3, various headings; Grant, 1930; La Motte et al_., 1932.) ILLUMINATION Illumination b y artificial light is superior to daylight for detailed study of protozoa. I n using high magnification objectives it will be found that reduction of the light source to a small area will give m u c h better results. Lamps should be provided with iris diaphragms. The lamp should be placed a foot or more from the microscope and the beam directed so as to strike the mirror. This can easily be done b y removing the ground glass and adjusting the position of the lamp so that a spot of bright light appears o n the mirror. The ground glass should then be replaced and the m i r r o r (plane side only) adjusted to illuminate the object. The condenser m u s t be m o v e d to its proper position, then the iris diaphragm of the lamp closed until the whole f i e l d is just illuminated. B y removing the eyepiece and looking down the tube of the microscope the illumination can be checked. To obtain the best results from high power oil immersion objectives, immersion oil should be placed b e t w e e n the condenser and the u n d e r side of the slide, as well as between the o b jective and the slide. If this is not done w h e n a n objective of numerical aperture greater than 1.00 is used, the full value of the objective cannot be obtained, and illumination is reduced. Dark-field illumination is valuable; for the study of living protozoa, particularly in bringing out flagella and filamentous extensions of the b o d y w h i c h are difficult to observe i n transm i t t e d light. The exact and critical adjustments necessary for oil immersion w o r k w i t h a h i g h aperture dark-field condenser are difficult to make. It is necessary that objectives of numerical aperture more than 1.00 be provided w i t h stops or iris diaphragms so that the effective aperture can be reduced. Dark-field illum i n a t i o n c a n be obtained b y placing u n d e r the substage a black metal disc with a central stop surrounded by a n opening, and adjusting the iris diaphragm;this is not adequate for critical h i g h power work. Phase m i c r o s c o p y is extremely valuable for study of structures i n living protozoa. Suit-

49

able phase objectives will reveal all that dark field does, w i t h the advantage of being easier to set up. In addition details of interior structure, w h i c h otherwise require staining, can be studied. It is possible with a n appropriate type of objective to make studies of certain lightly stained or faded m o u n t e d preparations that otherwise w o u l d have to be restained.

IMMOBILIZATION To study in detail the structure of active protozoa, their movements must be restrained. There are a number of ways of bringing this about without killing the organisms. If some ciliates, as Paramecium, are m o u n t e d w i t h b a c terial zoogloea under a cover glass (which should be rimmed with vaseline or paraffin) m a n y of them will eventually come in contact w i t h the debris and remain quiet for a time. Shaking by hand or in a mechanical device for several minutes will slow down or arrest the movement of Paramecium caudatum (King and Beams, 19^1). Centrifuging at moderate speed for a time has a similar effect. Use of methyl cellulose has b e e n found to have m a n y advantages (Stiles and Hawkins, 19^7)• A solution of Methocel (15 centipoises viscosity rating) is prepared as follows. A 10 gram amount is m i x e d in 4-5 cc. of w a t e r at boiling temperature, and the mixture allowed to stand 20 to 30 minutes; then an equal amount of cold water is added and the mixture stirred after cooling until smooth. A ring of the solution is m a d e o n a slide, the ring being of size suitable to be covered by a cover glass, a drop of water with the organisms is placed inside the ring, and the cover glass is added. Slowing down occurs progressively as the methyl cellulose diffuses inward. Movements m a y also be retarded b y narcotics. A small amount of saturated sodium amytal m a y be added and allowed to act f o r 20 to 30 minutes. Other anaesthetizing agents are 10$ methyl alcohol; Rousselet solution (cocaine 2$ aqueous, 3 cc; alcohol 90$, 1 cc; distilled water, 6 cc.); Corri solution, 96$ methyl alcohol, 10 cc.; w a t e r (salt or fresh), 90 cc.; chloroform, 3 drops; and 33$> magnesium sulphate.

IODINE Iodine is u s e d for staining GLYCOGEN and other substances, as a fixative in f l u i d foim and vapor, for washing out mercuric chloride following fixation, for staining flagella, and f o r staining of temporary preparations of cysts of amoebae. F i x a t i o n b y iodine vapor is accomplished b y adding a few drops of 95$ alcohol to some crystals of iodine in a small container, covering w i t h glass and heating to 30°C. and exposing the preparation to the vapor for from one-half to one minute for small flagellates. W a s h in water.

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA F o r fixation and staining Lugol iodineiodide solution and Gram solution are used. LUGOL SOLUTION is water 100 cc., potassium iodide 6 grams, iodine 4 grams. GRAM SOLUTION is water 300 cc., potassium iodide 2 grams, iodine 1 gram. As a killing agent and fixative, Lugol solution is added to the water containing the protozoa alone or combined w i t h some other fixative. F o r washing out mercuric chloride, a small amount of Lugol solution or of Gram solution Is added to 70$6 alcohol until it has a light, translucent b r o w n color. After fixation, the preparations are washed in water or clear 50$ alcohol and placed in iodine alcohol 10 minutes. F o r staining cysts of amoebae in temporary preparations a n iodine solution or D'Antoni iodine stain is used. The stain is made up b y adding 1.5 grams of powdered iodine crystals to 100 cc. of 1$> potassium iodide. D'Antoni (1937) recommended preparation of the stain b y special standardized methods. J.S.B.STAIN F O R B L O O D PARASITES. Manwell (19^5) stated that this method is the best w h e n both rapidity and high quality are desired, and it is easy to use. Solution I Methylene Blue (medicinal) Potassium dichromate sulfuric acid Water 1% potassium or sodium hydroxide solution

0.5 0.5 3.0 500

gram gram cc. cc.

10 cc.

Dissolve dye, add aci 24 A p a t h y c e m e n t , f o r s e a l i n g , 53, 56 A q u a t i c i n v e r t e b r a t e s , a t t a c h e d p r o t o z o a o n , 6,

8, 20

A q u a t i c p l a n t s , c i l i a t e s on, 6, 14 A q u e o u s m o u n t s , s e a l i n g , 56 Arcella, cultivation, 5 A r c h e t t i , 19 A r t h r o p o d s , h o s t s of p r o t o z o a , 18, 20, 2 3 , 27 A s c l e p i a d a c e a e , f l a g e l l a t e s in, 22 A s p i d i s c a , as f o o d f o r E u p l o t e s , 10

Astasia-type flagellates, in Turbellaria, A u t o t r o p h i c f l a g e l l a t e s , l i g h t f o r , 11

21

Baas-Becking, 9 B a b e s i a , 26 Bacillus subtilis, 5 B a c t e r i a : as f o o d f o r c i l i a t e s , 5, 6, 1 0 , 15; f r e e i n g p r o t o z o a f r o m , 2 - 3 , 24 B a c t e r i a - e a t i n g p r o t o z o a , 5» 6, 10, 1 1 , 1 2 , 14, 15 B a c t e r i a l z o o g l o e a , 31 B a c t e r i u m p r o d i g i o s u m , 18 B a c t o - B e e f , u s e i n b l o o d a g a r , 1.9 B a c t o - P e p t o n e , i n b l o o d a g a r , 19 B a l a m u t h , 20-21 B a l a n t i d i u m , c u l t i v a t i o n , 1 8 , 2 0 , 21; o c c u r r e n c e , 20 B a l s a m : f o r m o u n t i n g , 52; f o r s e a l i n g , 53> 56 B a r k e r m e d i u m , f o r m a r i n e d i n o f l a g e l l a t e s , 5, 7 B a r n a c l e s , h o s t s of g r e g a r i n e s , 26 Bauer's method, in staining glycogen, 45 B e a m s , 49 B e e f e x t r a c t , 3, 11 Beers, 7 B e e t l e g r u b s , c o n t a i n i n g f l a g e l l a t e s , 21 B e e t l e s , h o s t s of g r e g a r i n e s , 2 6 - 2 7 B e l a r , 4, 5, 11> 17 B e l l i n g ' s i r o n a c e t o - c a r m i h e , 31 Benecke agar, 3 B e n e c k e s o l u t i o n : 3 , 5, 1 3 , 14; p r e p a r a t i o n o f , 5 B e n o i t f l u i d , 39 B e n s l e y , 45, 55 B e r r e b i , 19 B e s t ' s c a r m i n e , 32, 45 Bicocoecids, 8 B i r d s , h o s t s of p r o t o z o a , 2 2 , 26 B l a c k h e a d d i s e a s e , 22 B l a s t o d i n i u m , 21 B l a t t a , 26 B l a t t e l l a , 26 B l e a c h i n g , 32, 58 B l o o d - A g a r m e d i u m , 19, 2 8 B l o o d e x a m i n a t i o n , 32 B l o o d f i l m s : d e h a e m o g l o b i n i z e d , 44; d e s t a i n i n g , 44; f i x i n g , 3 2 , 44; m a k i n g o f , 3 2 , 44; s t a i n ing of, 32, 4 4 , 5 0 , 60; t h i c k , 3 2 , 44; t h i n , 3 2 , 44; w e t f i x a t i o n , 42 Blood protozoa: enumeration of, 39; examination f o r , 23 B l o o d - R i n g e r m e d i u m , 19 B l o o d - s e r u m m e d i u m , 19 B l o o d s e r u m , d r i e d a n d g a s t r i c m u c i n m e d i u m , 22 B l u e - g r e e n a l g a e , 11, 13 Bodian technique. See Protein silver B o d o , f i x i n g of, 43 B o d o n i d f l a g e l l a t e s , 3, 5, 21 B o g s a n d s w a m p s , as a m o e b o z o a h a b i t a t s , 4

[65]

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MATERIALS AND METHODS IN THE STUDY OP PROTOZOA

Borax-carmine: Grenacher's alcoholic, 34; precipitated, 34 Borrel stain, 33 Bouillon, 11 Bouillon agar; preparation, 11; use, 3, 4, 13 Bouin fluid, 39, 42 Bouin-urea, 42 Bottom-samplers, 9 Box-elder bug, host of flagellates, 22 Brandwein, 4, 8, 10 Brasll-Duboscq fluid, 39, 42, 46 Brasil's alcoholic Bouin fluid, 3 9 , 45 Brine protozoa, cultivation, 9 Buffer solutions, 33

Chlamydophrys, 5 Chlorogonium, 13 Chloromonad flagellates, 6 Chloromyxum, 18 Chlor-zinc-iodine, 34 Choanoflagellates, 8 Chondriosomes, demonstration of, 34-35, 42, 63 Chonotrichs, occurrence, 20 Chrysomonad flagellates, 3, 6, 8 Cilia staining: in permanent preparations, 35, 3 6 , 5 6 ; in water, 35-36, 54 Ciliates: concentration and cleaning, 12, 36; cultivation, 5, 6, 20; isolation from bacteria, 2-3; isolation from other protozoa, 2; occurrence and collection, 6, 19-20; relief Cabbage, use in culture media, 7 staining for surface structures, 56; staining Caecum of rodents, source of protozoa, 18, 21, of cilia, 35-36, 54; technique of prepara22, 23 tion, 31, 35, 54-55 Calkins, 12 Citric acid, 6 Calonympha, 22 Claff, 2, 3 Camera lucida: 33, 37; enlargement of image, 33} Clarite: for mounting, 52;- for sealing preparaused for measuring, 52 tions, 56 Camp, 12 Clark, 48 Canada balsam, for mounting, 52 Cleaning slides and glassware, 2, 36 Capturing protozoa: by plankton net, 1, 6, 7, 8, Clearing, 36 13; on immersed slides or cover glasses, 1, Cleveland, 24, 25 14; on immersed Syracuse dishes, 1 Clorox, 37 Carbol-xylol, 36 Coccidia: occurrence, 25-26; oocyst flotation, Carbon pencil technique, 37 37; oocyst sporulation, 59 Carmine, alcoholic chlorhydric, 34, 55 Coccidiomorpha, 25-26 Carmine, Best's, 32, 45 Cockroaches: hosts of protozoa, 18, 19, 21, 22, Carmine staining, 33-34, 55 24-25, 26; maintenance of, 27 Carnoy fluid, 39, 42 Coelosporidium, 18 Carnoy-Lebrun fluid, 42 Collection and cultivation methods: for freeCattle, hosts of protozoa, 20, 26 living protozoa, 1-15; for symbiotic protoCattle ciliates, cultivation, 20 zoa, 18-28 Cat3, hosts of protozoa, 22, 26 Collection methods, general, 1, 23 Cellulose, tests for, 34 Collier, 24 Centipedes: hosts of gregarines, 26; maintenance Co.lpodid ciliates, 6 of, 27 Commercial fertilizer, 6 Centrifuging: concentrating organisms by, 3 6 ; to Concentration and cleaning of protozoa, 8, 12, slow movement, 49 36, 37 Cepedea, cultivation, 25 Concentration of cysts, 36-37 Cephalopods, hosts of Aggregata, 26 Conchophthirus, 20 Cercobodo, 42 Conejos, 28 Cercomonads, 3 Conjugation and nuclear reorganization in ParaCereal medium, 5 mecium, 12 Chaetogaster, host of flagellates, 21 Copepods, hosts of protozoa, 14, 18, 20, 21 Chalkley solution, 4, 10, 12 Copper salts, for shape preservation, 37, 54, 56 Champy fluid, 40, 42, 63 Copper sulfate, 37, 56 Champy-Kull technique, 35 Coprophilic protozoa, 3, 6 Champy-Regaud technique, 35 Coria, 18 Chatton, 40 Coronympha, 22 Chatton-Lwoff silver impregnation technique, 58 Costia, 21 Chen, 10, 31, 39, ^2 Cotton blue, 56 Chemical formulas of inorganic compounds in cul- Counterstaining, 34, 47, 55 ture media, 3 Cover glasses: cleaning of, 3 6 ; flotation of, 1; Chickens, hosts of protozoa, 22, 26 preparing blood films on, 32-33; thickness, Chicks, use in maintaining intestinal protozoa, 50 24 Cowdry, 55 Chilodonella, 10 Cow manure, in culture medium, 14 Chilomastix, 21 C.P.L.M. medium for Trichomonas, 27 Chilomonas: cultivation, 5-6; food for protozoa, Crabs, hosts of protozoa, 26 4, 5, 6, 7 , 10, 13, 14 Craig, C.F., 19, 22, 23 Craig, G.M., 24 China blue, 56 Crane-fly larvae, hosts of protozoa, 21, 23, 26 Chlamydomonas, 6, 13

MATERIALS A N D METHODS IN T H E STUDY OP PROTOZOA Cresyl blue, 32, 60 Crithldia, 22 Cryptobia, 21 Cryptocercus, host of protozoa, 22, 26, 59 Cryptomonad flagellates, 3 Cull, 12 Cultures, light for, 11 Culture types, 1 Culture vessels, 1-2, 12 Cyathomonas, 42 Cycloposthiidae, 20 Cyclops: 18; euglenoids attached to, 21 Cyst formation, In colpodid d i l a t e s , 6 Cysts, concentration of, 36-37 D'Antonl Iodine stain, 31, 50 D a Fano fixative, 40 Daphnia, euglenoids attached to, 21 Dark-field illumination, 49 Dasytricha, 20 Davis, 59 Deflandre, 14 De Freitas, 47 Delafield haematoxylin: use, 48, 55; formula, 48 Depression slides, 2, 12 Dero, host of coccidia, 26 Deschiens, 24 De Tomasi, 39 Devescovinid flagellates, 22 De V o l t , 22 Diatoms, 4, 5, 6, 7, 8, 9 Didinium, 7 Dimastigamoeba, cultivation and study, 7 Dlnoflagellate, plates, 37 Dinoflagellates: collection, J; cultivation, 3, 5, 7; symbiotic, 21 Dioxan, for clearing, 36 Diplocystis, 26 Dobell, 48 D o g s , hosts of protozoa, 22, 26 Drawing, 37 Drbohlav formula, Ringer, 25 Duco cement, for sealing, 56 Dunaliella, 7, 9 Earthworms: hosts of protozoa, 23, 26, 45; m a i n tenance of, 26 Earwigs, hosts of gregarines, 26 E a u de Javelle, 37 Eelgrass, foraminifera on, 9 Egg slants, 20 Egg yolk media, 5, 7, 14, 15, 20-21, 22, 23 E h r l i c h acid haematoxylin: formula, 48; use, 55 Eimeria, 25, 26 Eimeriidea, 2 5 Endamoeba, 18, 19 Endolimax, 18 Entamoeba, 18, 19, 23, 31 Entosiphon, 11 Enumeration of net plankton catch, 37-38 Enumeration of organisms, 38-39 Enumeration of protozoa in blood, 39 Ephestia, host of gregarines, 27 Erlicki, fixative, 42 Eudiplodinium, 20 Euglena: 2, 6, 7 - 8 , 13, 15; cultivation of, 7 - 8 , 15

67

Euglenamorpha, 21 Euglenoid flagellates, cultivation of, 3> 6, 8, 11 Eugregarinida, 2 6 Euparal, for mounting, 52, 53 Euphorbiaceae, flagellates in latex, of, 22 Euplotes: cultivation of, 10; staining of, 35, 36 Excystation of ciliates, 6, 7 Exuviaella, 7 Past green, for counterstaining, 55 Paure-Fremiet, 57 F a t : osmic test for, 51; staining tests for, 51 Fecal extract medium, 20, 21 Fecal examination: concentration methods, 36-37; coprophilic protozoa, 6; iodine staining, 31-32, 49-50; nuclear 3taining i n temporary preparations, 53 Feeding of cultures, 8-13 F e u l g e n nucleal reaction, 33, 39» 45, 5 5 Finley, 15 P i s h : hosts of protozoa, 6, 18, 21, 22, 23, 25; use in feeding cultures, 8 Fixation: b y formalin, 43; b y iodine vapor, 49; b y osmic vapor, 4l; time and temperature, 42 F i x a t i o n fluids: formulas, 39-42; mixing from stock solutions, 41-42 F l a g e l l a staining and demonstration, 49, 51> 56, 59 Flagellates: mass cultivation, 6; occurrence and collection, 8, 21-22; technique of preparation, 42-43 Flasks for mass cultures, 2 Pleas, hosts of coccidia, 26 Flemming fluid, 40, 42 F l o t a t i o n methods, 36-37 F l o u r - h a y medium, 9 Flour, use in feeding cultures, 8 F l u i d m e d i a f o r covering slants, 25 P o n e r , 18 P o o d vacuoles, 54 Foraminifera: occurrence and collection, 9; technique of preparation, 43 Formaldehyde, 43 Formalin: f o r fixation, 40; for preservation, 43 Fossil foraminifera, 9 Fossil radiolaria, 13-14 Fossil silicoflagellates, 8 Frogs, hosts of protozoa, 18, 19, 20, 21, 22, 25 Gall bladder, for amoebosporidia, 18 Galleria, host of schizogregarines, 27 Gammarid crustacea, protozoa on, 20 Gammarus, microsporidia in, 18 Ganapati, 28 Gastric m u c i n and dried blood serum m e d i u m , 22 Gastrotrichs, hosts of flagellates, 21 Gelei g o l d impregnation method, 57 Gelei gold technique, 57 Gelei-Horvath silver method, 57 Gelei modification of K l e i n technique, 57 Gelei sublimate-dichromate-alum fixative, 40, 42 Gelei tannin-silver technique, 57 Gersh, 55 Giardia, 22 G i e m s a staining; dry, 43-44; wet, 44

68

MATERIALS A N D METHODS IN THE STUDY OP PROTOZOA

Giese, 12 G i l s o n fixative, 42 Glaser, 18 Glycerine, f o r mounting, 52 Glycerine jelly, 37, 44-45, 52, 53 Glychrogel, 45 G l y c o g e n staining, 45 Golgi bodies, 45 Gonium, 14 G r a m solution, 45, 50 Grasshoppers, hosts of gregarines, 26 G r a y : rapid preparation methods, 55; recommendations for stock solutions, 41-42 Gregarines: fixation of, 45-46; occurrence and collection, 26-27, 45-46; occurrence and preparation of cysts, 45-46 Gregarinina, 25-26 Grenadier's alcoholic borax carmine, 34 Ground squirrels, hosts of flagellates, 21 Grouse, hosts of flagellates, 22 G u i n e a pigs, hosts of protozoa, 21, 22, 26 G u m arable and syrup, for mounting, 52 G u m arable, for mounting, 52 Gum chloral, for m o u n t i n g , 52 G u m damar, for mounting, 52 Gum tragacanth, for mounting foraminifera, 43 Gymnamoebae, 5 H a e m a t e i n staining, 47 H a e m a t o x y l i n crystal violet, 53 H a e m a t o x y l i n solution: adjustment of pH, 46; alcoholic, 46; ripening, 46, 47; stock, 46 H a e m a t o x y l i n staining: 46-48; alum haematoxylin technique, 47-48; Heidenhain technique, 46-47 Haemogregarines, occurrence, 26 Haemogregarinidea, 26 Haemoproteus, 26 Haemosporidea, 26 Hahnert solution, 9, 13 H a l l , 39 Hammond, 10, 59 Haplosporidia, 18 Harding, 33 Harris, 6 Harvey, 24 Hawkins, 49 H a y infusion: 5, 6, 7, 9, 10, 12; preparation,

9-10

Hegner, 24 H e i d e n h a i n m e t h o d of iron haematoxylin staining. See Iron haematoxylin, 46-47 H e i d e n h a i n Susa fixative, 40, 42 Heliozoa, 10 Helix, host of protozoa, 21, 26 Hemacytometer, use in counting, 39 Hemiptera: hosts of coccidia, 26; hosts of f l a g e l l a t e s , 21, 22 H e n d e r s o n , 24 Henneguya, 18 Hepatozoon, 26 H e r m a n n fluid, 35, 40, 42 Herpetomonas, 22 Hetherington, 2, 13 H e t h e r i n g t o n 1 s m e d i u m for Tetrahymena, 15 Hewitt, 32, 36 H e x a m i t u s , 22

Hirschler fluid, 40, 42 Hirschler technique for Golgi, 45 Histomonas meleagridis, 22 Hitchcock, 20 Hogue, 22 Hollande: 11, 32, 35; o n fixing flagellates, 42 Hollande cupric picro-formol fixative, 40, 42 Holothurians, hosts of protozoa, 23 Honigberg, 47, 58 Horses, ciliates in caecum and colon, 20 H o r v a t h silver technique, 58 Host examination procedure, 23 Hungate, 20 Hydra, 20 Hydroids: hosts of protozoa, 23; suctoria f o u n d on, 20 H y d r o g e n - i o n concentration: changes in food vacuoles, 54; determination of, 48-49 Hyman, 11, 12 • Hypermastigote flagellates, 22 Hypotrichs: 1, 6; cultivation, 10, 15 Ichthyophthirius, 20 Illumination in microscopy, 49, 50 Immersed slides and cover glasses, for capture of protozoa, 1, 14 Immersed Syracuse dishes, for capture of protozoa, 1 Immobilization, 49, 54 Indicator dyes, for pH, 48-49 Insects, peritrichs on, 20 Insects: protozoa in, 18-19, 20, 21, 22, 23, 24-25, 26-27; suctoria on, 20 Indulin, for counterstaining, 55 Intercellular connections, staining of, 34 Intestinal flagellates and amoebae, cultivation, 23-24 Invertebrates, marine, hosts of protozoa, 23 Iodamoeba, 18 Iodine staining, 45, 49-50 Iodine vapor fixation, 43, 49 Iron haematein staining, 46, 47 Iron haematoxylin, counterstaining after, 47 Iron haematoxylin staining: differentiation of stain, 46-47; fixing of material for, 46; laboratory schedule for, 47; short cuts, 47; technique, 46-47; use, 31, 42, 43, 55, 59; variations of method, 46-47 Isolation from bacteria, 2 - 3 , 24 Isolation technique, 2-3 Isospora, 26 Isotricha, 20 Jacobs, 21 J a h n , 24 Janus G r e e n B , 34, 60 Jennings, 12 Jirovec, 22, 27 Johnson, 24, 27, 39 Jones, 36 J.S.B. stain for b l o o d parasites, 3 2 , 50 Kalotermitidae, 22 Karotomorpha, 21 Karyolysus, 26 K a t e r , 13

MATERIALS A N D METHODS IN THE STUDY OP PROTOZOA K a y , 10 K e l s e r , 19 K e r o n a , 20 Khawkinea, 21 Kidder, 2 Killing protozoa: b y copper salts, 37; b y osmic vapor, 40-41, 5^ King, 49 Klebs solution, 8, 10 K l e i n silver nitrate method', 57 Klossiella, 26 K n o p agar, 3, 5, 6, 8, 11, 13 K n o p solution, 3, 6, 7 , 10, 13, 14 Kofoid, 23, 24 Kolatchev technique, 42, 45 Konsuloff, 25 K u d o , 9, 12-13 Lackey, 1, 11 Lackey wheat medium, 5> 11 Langeron description of heart puncture methods, 32 Lankesterella, 25 Lanoline resin, for sealing preparations, 53, 56 Laudermilk, Lee, 39 Lefevre medium,8, 11 Leishmania, 19, 24 Leishman stain, 32, 50-51 Leptomonad flagellates, 22 Leptotheca, 1 8 Lettuce infusion, 11, 12 Lettuce, use in feeding cultures, 8, 11 Leucocytozoon, 26 Light green, for counterataining, 55 Light, in growth of protozoa, 11 Lipids, staining tests for, 51 Liquid media, to cover slants, 23, 24 Little, 28 Liver infusion agar, 23,.24, 27 Lizards, hosts of protozoa, 21 Locke-blood medium, 24 Locke-egg-blood medium, 27 Locke-egg medium, 22 Locke solution, 18, 24 Loefer, 39 Loeffler dehydrated beef serum, 25 Loeffler dried blood serum, 22, 24, 27 Loeffler stain for flagella, 51 Lophomonas, 22, 24-25 Lorenc, 24 Low temperature preservation, 25 Ludford technique for Golgi, 45 Lugol solution: 31, 43, 50, 59; preparation, 59 Lumbricus, 26 Luminescence, of dinoflagellates, 7 Lwoff, 25, 40, 50 L y n c h precipitated borax carmine method, 31 McClung, 34 Mcllvaine citric acid phosphate buffers, 33 M c N e i l , 23, 2 4 Macroscopic camera lucida drawing apparatus, 33 M a g n i f i c a t i o n of camera lucida drawing, 52 Mahmoud, 24 Mainx, 8

69

M a l t e d milk, use in culture media, 8 Mammals, hosts of protozoa, 18, 19, 20, 26 M a n , host of protozoa, 18, 22, 2 6 M a n n methyl blue eosin stain, 43, 51 Manwell, 25, 50 Mastigina, 21 Mating type reaction in ciliates, 12 M a y e r albumen fixative, 31, 51 M a y e r haemalum, 48 May-GrUnwald solution, 44 Mead's cereal, 5 Mealworms: hosts of gregarines, 2 6 ; maintenance of, 26, 27 Measurement, 52 Meat extract, 3, 4, 11 Meat extract agar, 3, 4, 7 Mechanical stage readings, 52 Menoidium, cultivation, 8 Merton, 55, 56, 57 Metacoronympha, 22 Methyl cellulose, 49 Methyl g r e e n acetic, 52, 53, 5^ Meyer, 28 M i c e , hosts of protozoa, 22, 26 Microincineration, 52 Microscope, care of, 50 Microscopy: illumination for, 49; laboratory procedure in, 50 Microsporidia: occurrence, 18, 23; spore filament discharge, 59 M i g r a t i o n m e t h o d s : in concentrating ciliates, 3 6 ; in freeing protozoa of bacteria, 2-3 M i l l o n reaction, 55 Mitochondria. See Chondriosomes. M o l e crickets, hosts of flagellates, 21 M o l i s c h solution, 11 Monocercomonoides, 21 Monocystids: fixation, 45; occurrence, 26, 45 Moore solution, 11 Morgan, 24, 27 Morgenstern, 39 Morse, 13 M o s s : habitat of amoebozoa, 4; use in culture media, 7 Mounting media, 44-45, 52-53 Movement retardation, 49 M u s c o i d flies, hosts of protozoa, 21, 22, 23 Musgrave and Clegg agar medium, 3 , 7 Mussels, mantle cavity as source of ciliates, 20, 23 Mycetozoa, occurrence and cultivation, 3) 11-12 Myers, 31, 43 Myxidium, 18 Myxobolus, 18 Myxosoma, 18 Myxosporidia: 18; spore filament discharge, 59 Naegleria gruberi, 7 Nelson, 20, 21, 23, 36 Neutral red, 32, 54, 60 Nigrelli, 21 Nigrosin, 54, 56 N i l e blue sulphate, 51 N i n h y d r i n reaction, 55 Nitzschia, 9 Nosema, 18

TO

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA

pH, determination of, 48-49, 54 Phase microscopy, 49 Pheasants, hosts of flagellates, 22 Phelps, 2 Phleger, 9 Phloxlne-rhodamine solution, 56 Phototropism, green flagellates concentrated by, 36 Physiological balanced solution, 5, 13, 15 Oatmeal: agar, 12; for feeding Mycetozoa, 12 Phytomonad flagellates, 3, 8, 13, 15 Octomitus, 22 Phytomonas, 22 Ocular micrometer: calibration of, 52; measurePicro-carmine, 34 ment by, 52 Picro-formol-acetic fixative, 39 Oil immersion objectives, use, 49, 50 Pietschmann, 7 Oligochaetes, hosts of protozoa, 20, 23, 26 Pipettes, for transfer of cultures, 2 Oliviera, 2.8 Piroplasmidea, 25 Oodinium, 21 Plankton animals, protozoa on, 14, 20 Opal blue, 56 Plankton: collection, 1, 6, 8, 13; enumeration, Opalina, 19, 25 37-38 Opalinid ciliates: cultivation, 25; occurrence, 25 Plants, flagellates occurring in, 22 Plasmodium: cold preservation, 25; occurrence, Ophryoscolecidae: cultivation, 20; occurrence, 26 20; skeletal plates stained, 34 Plistophora, 18 Oscillatoria, 13 Osmic acid, 40-4l Polychaete worms, hosts of schizogregarines, Osmic vapor fixation, 4l 26, 27 Osmic vapor, use In killing, 7 Polymastix, 21 Osmium fixatives, washing after, 40 Polytoma, 13 Osterhout, 13 Polytoma medium, 13 Otto, 36, 53 Polytomella, 13 Oxyrrhis, 5, 7 Post, 34 Oxytricha, 10 Powell, 27 Preservation by low temperature freezing, 25 Pringsheim, 5, 8, 11, 14 Pandorina, 6, 13 Protargol. See Protein silver. Parabasal body, demonstration of, 59 Parafilm, used to cover culture tubes, 27 Protein silver impregnation: bleaching process, Paramecium: 2, 3, 7, 8, 11, 12, 15, 35, 42; 58; fixation for, 58; procedure, 58-59; use, avoiding reaction in, 53; collection, 12; 43, 54-55, 59-60 conjugation, 12; cultivation, 2, 3, 7, 12; Protein reactions, 55 immobilization, 49, 54; killing for observaProteomyxid rhizopods, 13 tion, 37; mating types, 12; methods of obProteromonas, 21 servation in aqueous mounts, 53; schedule for Pseudomonas, 5 preparation, 55; shape preservation, 37, 56; Protozoa: collection of, 1, 4, 5, 6, 7 , 8, 9, staining of cilia, 35, 54; study of food cur11, 12, 13, 14, 18, 19, 21, 22, 23, 25, 26; rents and ingestion, 54; study of locomotion, concentration of, 8, 12, 36-37 53; technique of preparation, 54-55 Purple bacteria, 6 Paulson, 39 Putter solution, 24, 25 Pavlova, 22 Pyrex water, 12 Pea infusion, 8, 10 Peat agar, 3, 7, 8, 13 Quail, hosts of protozoa, 22, 26 Peat extract, 6, 12, 13 Quensel's stain, 32, 53 Pelomyxa, cultivation, 9, 12-13 Penard, 4 Rabbits, hosts of protozoa, 22, 26 Pencils, kinds for drawing, 37 Radiolaria: 8, 13-14; fossil, 13-14 Penicillin, for bacteria-free culture, 3, 24 Radiolarian ooze, 13 Pens, kinds for drawing, 37 Rafalko, 7 Pentatrichomonas hominis, 22, 24 Rana, 25 Peptone, solution, Arlco, 13 Rapid preparation of permanent stained mounts, Pentatrichomonas, 22, 24 55 Peranema, 5 Rats, hosts of protozoa, 21, 22, 23, 26 Perenyi fluid, 4l Rawson, 20 Perezia, 18 Reardon, 24 Periplaneta, 26 Rees, 24 References on collection and cultivation of freePeritrichs, occurrence, 6, 20 living protozoa, 15-17 Peter Gray sealing medium, 56 References on collection and cultivation of Peters medium, 5, 13, 15 symbiotic protozoa, 28-30 Petrunkevitch fluid, 4l, 42 Nuclear reorganization in Paramecium, 12 Nuclear staining: in permanent preparations, 33-34, 39, 43, 44, 51; in temporary preparations, 53 Nutrient agar: use, 5, 18, 19; preparation, 3, 19 Nyctotherus: cultivation, 20; occurrence, 20

MATERIALS AND METHODS IN THE STUDY OP PROTOZOA References on technical methods, 60-63 Regaud fixative, 63 Regaud haematoxylin, 35, 40, 63 Reiehenow, 19 Relief staining, 56 Reptiles, hosts of protozoa, 18, 19, 21, 26 Reticulitermes, 22 Retortamonas, 21 Rhizomastigina, 21 Rhizopods: collection, 4-5, 18-19; cultivation, 3, 5, 11, 12, 13, 23-24; preparation, 31-32 Rice grains, use of, 8, 10, 12-13 Rice infusion, 8, 10 Rice stalks,. 10 Rice starch, 18, 20, 21, 22, 24, 27 Rieder, 14 Ringer and horse serum, 24 Ringer solution, 18, 22, 25 Rio Ortega ammoniacal silver carbonate, 58 Rodents, as hosts of protozoa, 18, 19, 21, 22,

23, 26

Rodova, 22, 27 Rolled oats, 11-12 Romanowsky stains, 44 Rous3elet solution, 49 Ross stipple board, 37 Rotifers, hosts of flagellates, 21 Ruinen, 9 Ruminants, ciliates in rumen of, 20 Saline serum culture medium, 22 Salt marsh pools, habitat of protozoa, 6, 7 Sampath, 28 Sanders, 24, 25 Sandon, 3 6 , 37 Sands, dinoflagellates found on, 7 Sandza, 20 Sarcocystis, 18 Sarcosporidia, 18 Schaudinn fluid, 4l, 42 Schiff reaction, 39 Schizogregarines, 26, 27 Schneider sodium citrate medium, 27 Scytomonas, 8 Sealing preparations, 53, 56 Sea water, synthetic, 4, 14 Seaweed, foraminifera on, 9 Sections, preparation of, 32, 42-43, 56 Sedgwick-Rafter method, 37 Selenidium, 27 Seneca, 24 Senekjie, 28 Sepia, 26 Serratia, 5, 18 Serum fluid media, 22, 27 Serum slants, 23, 25 Shape preservation, 37, 56-57 Shelled rhizopods, 4, 5 Shih Lu Chang, 28 Silicoflagellates, 8 Silver impregnation: techniques, 54, 57-59; use, 43, 54 Slime molds, 11-12 Snails, hosts of protozoa, 21, 26 Snakes, hosts of haemogregarines, 26

71

Sodium amytal, for retarding movement, 49 Soil-cheese medium, 5, 8, 17 Soil extract, 6, 8, 13, 14, 15 Soil protozoa, 14 Sonneborn, 12 s/rensen phosphate buffers, 24, 33 Specht, 14 Sphaeromyxa, 18 Sphagnum extract, 12 Sphagnum, habitat of thecamoebae, 4 Spirillina, 43 Spirostomum, cultivation, 14 Spirostomum, shape preservation, 37, 56 Spirochonids, 14 Sponge growths, foraminifera on, 9 Spore filament discharge, 59 Sporozoa, occurrence, 25-27 Sporulation of oScysts, 59 Spring water, artificial, 10 Stage micrometer, 52 Stained mounts, rapid preparation of, 55 Staining: after osmic fixatives, 43; Borrel technique, 33; carmine, 33-34; cilia and flagella, 35-36, 51, 54; dry blood films, 44, 50, 51, 60; Peulgen nucleal reaction, 39; for nuclei in aqueous mounts, 31, 49-50, 52, 53, 54; haematoxylin, 46-48; methyl blue eosin, 51; vital and supravital, 60; wetfixed preparations by Giemsa, 44 Stain tests: for cellulose, 34; for glycogen, 32, 45; for fat, 51; for protein, 55; for starch, 59; for volutin, 60 Starch: in culture, 27; test for, 59 Stenostomum, 10 Stempellia, 18 Stentor: cultivation, 14, 15; shape preservation, 5 6 , 57 Stentorids, 6 Stephanonympha, 22 Sterilization technique, 2-3, 20, 24 Stiles, 49 Stock solution: for fixation fluids, 41-42; of Giemsa stain, 43-44; of haematoxylin, 46 Stone, 24, 25 Stool examination. See Fecal examination. Strahan, 36 Streptomycin, 24 Stylonychia, 10 Subbarow, 28 Sublimate acetic, 4l Suctoria, collection and occurrence, 14 Sudan dyes, 51 Syndlnium, 21 Synthetic sea water, 4, 14 Synthetic spring water, 14 Syracuse watch glasses, 1, 2 Syrup of Apathy, 53 Tadpoles: Euglenamorpha In intestine of, 21; hosts of flagellates, 21, 22 Taylor, Lois C., on drawing, 37 Taylor and Strickland, 13, 15 Technical methods of study and preservation, 31-60 Tenebrio: laboratory source of gregarines, 2 6 ; maintenance, 26

72

MATERIALS AND METHODS IN THE STUDY OF PROTOZOA

Termite flagellates: cultivation, 27; preparation of, 59 Termites: hosts of protozoa, 21, 22, 27, 59; removing gut of, 59 Tetrahyraena geleii, 15 Theca, of armored dinoflagellates, 37 Thecamoebae: occurrence, 4, 5; preparation for tests, 32 Theileria, 26 Thelohania, 18 Thigmotrichs, in mussels, 20 Thompson, 25 Thymonucleic acid, test for, 39 Tillina, 6 Tintinnids, 6 Toads. See Amphibia. Trager, 2, 27 Tretomphalus, 43 Tricercomitus, cultivation, 27 Trichocysts, study of, 54 Trichodina, 20 Trichomonad flagellates: cultivation, 22, 27; occurrence, 22, 27; preparation of, 59 Trichomonas foetus. See Tritrichomonas foetus. Trichomonas hominiB. See Pentatrichomonas hominis. Trichomonas vaginalis, cultivation, 24, 27 Trichonympha, 22, 27 Tritrichomonas, in amphibia and mammals, 22 Tritrichomonas foetus, cultivation, 27 Trussell, 24, 27 Trypanosoma: cultivation, 19, 28; in blood of vertebrates, 21-22; preservation by freezing, 25 Trypanosomatidae, 19, 21, 28 Tubifex, 18 Tungstic haematoxylin staining, 48 Tunicates, hosts of schizogregarines, 26 Turbellaria, hosts of flagellates, 21 Turkeys, hosts of flagellates, 22 Turner, 10 Urinary bladder, for amoebosporidia, 18 Uspenski and Uspenskaja, 15 Velat, 53 Vital and supravital staining, 60 Vital stains, use of, 32, 54, 60

Volutin, 60 Volvox, cultivation, 15 Vorticella, cultivation, 15 Vorticellids, shape preservation, 56-57 Wagner, 24 Watch glasses, 2 Water plants, attached d i l a t e s on, Water samples, 1 Watson, 6 Wax moth, 27 Weinman, 28 Weinstein, 53 Wenrich: 22, 27, 4l, 42; concerning fixation of protozoa, 42; medium longed cultivation of intestinal 22, 27 Wettstein, v., 3, 5 Wilcox, 44 Willis, 37 Wood-boring beetle larvae, hosts of 23 Wheat grains, 4, 5, 8, 10, 12, 14 Wheat Infusion, 4, 5, 10, 14, 15 Whipple micrometer, 38 Wlchterman, 2, 3, 11, 12, 15 Wilber, 9 Wild cultures, 2 Wilson, 7 Wilson liver concentrate, 21 Wolff carbon pencils, 37 Wood roach, flagellates in, 59 Worcester fluid, 4l Wright stain, 32, 60

6

factors in for proflagellates,

protozoa,

Xanthoproteid reaction, 55 Yeast, dried, 10 Yeast medium for excystation, 15 Yeast, used in culture, 25; for feeding Mycetozoa, 11 Yocum fluid, 4l, 42 Zenker fluid, 4l, 42 Zootermopsis, 22 Zoothamnium, shape preservation, 57 Zschokkella, 18 Zumstein medium, 8, 15