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SYNOPSIS. A classification of the phylum Protozoa at supra-familial levels is given with definitions or descriptions of all of the involved taxa, some 140 in number. The scheme represents the result of the cooperative efforts of an international group of specialists and consultants who have‘been studying the overall problem for the past several years. Innovations of a nomenclatural nature have been held to a minimum, aside from the use of a system of uniform endings, but several major shifts of taxonomic significance have been introduced at all levels treated, including the subphyla. These revisions are explained in appropriately placed footnotes.

In the first of these reports (Wenrich, 1946), it was pointed out that the cultivation of intestinal flagellates was originally undertaken as an aid in the study of trichomonad morphology. Two additional objectives have developed during the course of the work: 1) to determine the length of time that various intestinal flagellates will survive in individual culture tubes if, when populations begin to decline, additional nutrients are supplied and water is added to maintain the quantity of fluid; and 2) to consider the correlation between the capacity to live under diverse cultural conditions and the ability to reach new hosts. The present account includes results obtained on flagellates from amphibians and reptiles up to the end of October, 1946. I am indebted to Mr. H. A. Walters for the opportunity to examine two pilot black snakes (Elaphe obsoleta) and a Mobile turtle (Pseudemys floridana mobilensis) ; to Dr. T. S. Hauschka for collecting specimens of the blue-spotted salamander (Ambystorna jeffersonianum) and the northern red salamander (Pseudotriton ruber) ; to Dr. R. M. Stabler for other amphibians; and to Miss Bernice Goldis, who, under the supervision of Dr. L. V. Heilbrunn, made determinations of pH and of freezing-point depressions. This study has been aided by a grant from the Research Fund of the University of Pennsylvania. The methods previously outlined have been employed. The chief basic fluids have been 1) Drbohlav's (1925) modified Ringer's fluid (R2) and 2) boiled pond water (W). Sometimes regular Ringer's (R1), with 0.8% NaC1, or various dilutions of it, have been used. The principal nutrients have been 1) gastric mucin (M), and 2) Difco Loeffler's dried blood serum (L), both commonly in a concentration of 0.2%. For initial inoculations these substances were dissolved in the fluids, but when added later they were frequently in the dry state, unless liquid was also to be added, in which case, they were dissolved, usually in distilled water. Additional nutrients employed will be referred to in the descriptions and tables; in the explanation of the latter the meanings of the other abbreviations may also be found. Longevity figures give the number of days that individual tubes remained positive. As in the previous paper, original cultures are indicated by a capital letter following a strain number. Subcultures are designated by a small letter after a transplant-generation number. For example, in the third line of Table 2, culture 1E/4a is the first tube (a) of the fourth subculture-generation from the fifth (E) tube among the original cultures from frog 1. Since the original cultures were inoculated from feces or intestinal contents, the bacterial flora of these cultures originated from the same source. The usual precautions were taken to prevent the entrance of organisms from the air.

According to the compilations of Walton (1941) and of Morgan (1944), Trichomonas augusta has been reported from 19 species in 7 genera of urodele amphibians, from 33 species in 9 genera of anurans, and from one species of lizard. Trichomonas batrachorum is also fairly common in amphibia, but has not been reported from so many kinds of hosts: 2 species in 2 genera of urodeles, 22 species in 7 genera of anurans, 2 lizards and one snake. From these lists one might assume that T. augusta is the more hardy species, and the more successful in adapting itself to different kinds of hosts. Yet, if one places a frog's rectal contents containing both species into some of the culture media commonly employed for trichomonad flagellates, T. batrachorum will grow in almost any of them while T. augusta usually does not grow at all or, if it does develop for a short time, it will soon be overwhelmed by the more rapidly growing T. batrachorum. This situation has been a challenging one and on several occasions I have assigned to graduate students the problem of trying to cultivate T. augusta. Until recently these efforts have not succeeded in maintaining this species in vitro for more than about 2 weeks. Unexpected success was attained in the winter of 1914 4 5.

The culture experiments to be described in these reports were originally undertaken as part of a study of the morphology of trichomonad flagellates. It seemed desirable to test the suggestion made in some accounts in the literature that under cultural conditions the morphology of such flagellates sometimes undergoes alterations which tend to invalidate distinctions among the species. For the species cultivated it can be said that specific characters remained constant during this study. In culture work on trichomonad flagellates, which apparently do not form true cysts, it is customary to examine cultures and to make transplants at frequent intervals. This method of handling is based upon the widely accepted idea that culture populations decline as a result of the accumulation of noxious waste products as well as to an exhaustion of food supplies. In an effort to save time and labor in the present study, attempts were made to prolong the life of individual cultures by adding nutrients from time to time, and distilled water to replace that lost by evaporation, instead of making so many subcultures. Some of the cultures so treated have remained positive for surprisingly long periods of time. Another aspect of the work concerns itself with host-parasite relations. Presumably a trichomonad flagellate that lives in vitro has advantages in reaching a new host over one that can live only with difficulty outside the host. An intestinal flagellate of a warm-blooded host that survives at room temperature has, theoretically, a better chance of reaching a new host than one that requires temperatures close to that of the host. An intestinal flagellate that can develop in cultures made by adding nutrients to water probably has greater advantages in getting to a new host than one that cannot live in water. These various considerations have determined the nature of the culture work undertaken. In this first report, results of culture experiments on Trichomonas hominis and on intestinal flagellates from various rodents, handled up to the end of June, 1945, will be described. I am indebted to Dr. M. M. Rothman for specimens containing Trichomonas hominis; to Mr. LeRoy Saxe for a culture of this species from a dog; to Mr. H. A. Walters for opportunities to examine a Syrian hamster (Cricetus auratus) and two pocket gophers (Thomomys bottae) ; to Sharp and Dohme, Glenolden, Pa., and to Dr. H. A. Shelanski, of Smyth Laboratories, Philadelphia, for guinea-pigs; to Dr. Shelanski for the rats from which cultures were made; and to Mr. M. Frumin for a small series of white mice. I am also indebted to the Cerophyl Laboratories, Kansas City, Mo., for a sample of cerophyl, and to the Wilson Laboratories of Chicago for their cooperation in securing a supply of gastric mucin.

In the first of these studies (Wenrich, 1946), attention was called to the persistence of certain intestinal flagellates in single culture tubes if nutrients and water were added as needed. Basic fluids were Drbohlav's (1925) modified Ringer's fluid (R2) Or pond water (W), but sometimes regular Ringer's (R1) or some dilution of this, was employed. The nutrients most used were gastric mucin (M) and Difco Loeffler's dried blood serum (L), both in concentrations of 0.2%. Various other nutrients occasionally employed are indicated in the table and in the text. In initial cultures, the mucin and serum were dissolved in the fluids, but additions to the cultures were frequently made with the powdered material. However, when both food and fluid were to be added the nutrients were usually dissolved, customarily in distilled water.

Although representatives of the genus Endolimax have been reported for a considerable number of vertebrate and invertebrate hosts (Wenrich, 1935), there are few records of such representatives in reptiles. The amoeba from the European lizard, Lacerta agilis, first noted by Hartmann and Prowazek (1907) (named Amoeba lacertae by Hartmann in a footnote) and studied more fully by Nigler (1909), and also the one from Lacerta muralis (captured near Naples) studied by Dobell (1914), obviously do not belong in the genus Endolimax. Their nuclear division stages are different and their mature uninucleate cysts resemble those of certain small free-living amoebae. Hartmann (1914) referred to Dobell's species as Amoeba (Vahlkampfia) dobelli. The "Endolimax reynoldsi" described by McFall (1926) from the American swift, Sceloporus undulatus, with its mature uninucleate cyst, resembles much more the species described by Ndigler and Dobell than it does Endolimax, and therefore probably does not belong to this genus. Sanders and Cleveland (1930), in their paper on Entamoeba terrapinae, mention finding a small amoeba "similar to Endolimax nana" (p. 271) in Chrysemys picta. Apparently no cysts were seen. This is the only reference to an Endolimax-like amoeba in turtles that we have found. The form seen by these authors is probably the same as that described below.