“deficiencies,” fragmentations, translocations, etc., of portions of a chromosome. These cases are making possible attacks on a number of genetic problems otherwise difficult of approach.

The transmuting action of X-rays on the genes is not confined to the sperm cells, for treatment of the unfertilized females causes mutations about as readily as treatment of the males. The effect is produced both on oöcytes and early oögonia. It should be noted especially that, as in mammals, X-rays (in the doses used) cause a period of extreme infertility, which commences soon after treatment and later is partially recovered from. It can be stated positively that the return of fertility does not mean that the new crop of eggs is unaffected, for these, like those mature eggs that managed to survive, were found in the present experiments to contain a high proportion of mutant genes (chiefly lethals, as usual). The practice, common in current X-ray therapy, of giving treatments that do not certainly result in permanent sterilization, has been defended chiefly on the ground of a purely theoretical conception that eggs produced after the return of fertility must necessarily represent "uninjured" tissue. As this presumption is hereby demonstrated to be faulty it would seem incumbent for medical practice to be modified accordingly, at least until genetically sound experimentation upon mammals can be shown to yield results of a decisively negative character. Such work upon mammals would involve a highly elaborate undertaking, as compared with the above experiments on flies.

From the standpoint of biological theory, the chief interest of the present experiments lies in their bearing on the problems of the composition and behavior of chromosomes and genes. Through special genetic methods it has been possible to obtain some information concerning the manner of distribution of the transmuted genes amongst the cells of the first and later zygote generations following treatment. It is found that the mutation does not usually involve a permanent alteration of all of the gene substance present at a given chromosome locus at the time of treatment, but either affects in this way only a portion of that substance, or else occurs subsequently, as an after-effect, in only one of two or more descendant genes derived from the treated gene. An extensive series of experiments, now in project, will be necessary for deciding conclusively between these two possibilities, but such evidence as is already at hand speaks rather in favor of the former. This would imply a somewhat compound structure for the gene (or chromosome as a whole) in the sperm cell. On the other hand, the mutated tissue is distributed in a manner that seems inconsistent with a general applicability of the theory of "gene elements" first sug

gested by Anderson in connection with variegated pericarp in maize, then taken up by Eyster, and recently reenforced by Demerec in Drosophila virilis.

A precociously doubled (or further multiplied) condition of the chromosomes (in "preparation" for later mitoses) is all that is necessary to account for the above-mentioned fractional effect of X-rays on a given locus; but the theory of a divided condition of each gene, into a number of (originally identical) "elements" that can become separated somewhat indeterminately at mitosis, would lead to expectations different from the results that have been obtained in the present work. It should, on that theory, often have been found here, as in the variegated corn and the eversporting races of D. virilis, that mutated tissue gives rise to normal by frequent "reverse mutation"; moreover, treated tissues not at first showing a mutation might frequently give rise to one, through a “sorting out" of diverse elements, several generations after treatment. Neither of these effects was found. As has been mentioned, the mutants were found to be stable through several generations, in the great majority of cases at least. Hundreds of non-mutated descendants of treated germ cells, also, were carried through several generations, without evidence appearing of the production of mutations in generations subsequent to the first. Larger numbers will be desirable here, however, and further experiments of a different type have also been planned in the attack on this problem of gene structure, which probably can be answered definitely.

1

Certain of the above points which have already been determined, especially that of the fractional effect of X-rays, taken in conjunction with that of the production of dominant lethals, seem to give a clue to the especially destructive action of X-rays on tissues in which, as in cancer, embryonic and epidermal tissues, the cells undergo repeated divisions (though the operation of additional factors, e.g., abnormal mitoses, tending towards the same result, is not thereby precluded); moreover, the converse effect of X-rays, in occasionally producing cancer, may also be associated with their action in producing mutations. It would be premature, however, at this time to consider in detail the various X-ray effects previously considered as "physiological," which may now receive a possible interpretation in terms of the gene-transmuting property of X-rays; we may more appropriately confine ourselves here to matters which can more strictly be demonstrated to be genetic.

Further facts concerning the nature of the gene may emerge from a study of the comparative effects. of varied dosages of X-rays, and of X-rays administered at different points in the life cycle and under varied conditions. In the experiments herein re

ported, several different dosages were made use of, and while the figures are not yet quite conclusive they make it probable that, within the limits used, the number of recessive lethals does not vary directly with the X-ray energy absorbed, but more nearly with the square root of the latter. Should this lack of exact proportionality be confirmed, then, as Dr. Irving Langmuir has pointed out to me, we should have to conclude that these mutations are not caused directly by single quanta of X-ray energy that happen to be absorbed at some critical spot. If the transmuting effect were thus relatively indirect there would be a greater likelihood of its being influenceable by other physico-chemical agencies as well, but our problems would tend to become more complicated. There is, however, some danger in using the total of lethal mutations produced by X-rays as an index of gene mutations occurring in single loci, for some lethals, involving changes in crossover frequency, are probably associated with rearrangements of chromosome regions, and such changes would be much less likely than "point mutations" to depend on single quanta. A reexamination of the effect of different dosages must therefore be carried out, in which the different types of mutations are clearly distinguished from one another. When this question is settled, for a wide range of dosages and developmental stages, we shall also be in a position to decide whether or not the minute amounts of gamma radiation present in nature cause the ordinary mutations which occur in wild and in cultivated organisms in the absence of artificially administered X-ray treatment.

As a beginning in the study of the effect of varying other conditions, upon the frequency of the mutations produced by X-rays, a comparison has been made between the mutation frequencies following the raying of sperm in the male and in the female receptacles, and from germ cells that were in different portions of the male genital system at the time of raying. No decisive differences have been observed. It is found, in addition, that aging the sperm after treatment, before fertilization, causes no noticeable alteration in the frequency of detectable mutations. Therefore the death rate of the mutant sperm is no higher than that of the unaffected ones; moreover, the mutations can not be regarded as secondary effects of any semi-lethal physiological changes which might be supposed to have occurred more intensely in some ("more highly susceptible") spermatozoa than in others.

Despite the "negative results" just mentioned, however, it is already certain that differences in X-ray influences, by themselves, are not sufficient to account for all variations in mutation frequency, for the present X-ray work comes on the heels of the determination of mutation rate being dependent upon tempera

ture (work as yet unpublished). This relation had first been made probable by work of Altenburg and the writer in 1918, but was not finally established until the completion of some experiments in 1926. These gave the first definite evidence that gene mutation may be to any extent controllable, but the magnitude of the heat effect, being similar to that found for chemical reactions in general, is too small, in connection with the almost imperceptible "natural" mutation rate, for it, by itself, to provide a powerful tool in the mutation study. The result, however, is enough to indicate that various factors besides X-rays probably do affect the composition of the gene, and that the measurement of their effects, at least when in combination with X-rays, will be practicable. Thus we may hope that problems of the composition and behavior of the gene can shortly be approached from various new angles, and new handles found for their investigation, so that it will be legitimate to speak of the subject of "gene physiology," at least, if not of gene physics and chemistry.

In conclusion, the attention of those working along classical genetic lines may be drawn to the opportunity, afforded them by the use of X-rays, of creating in their chosen organisms a series of artificial races for use in the study of genetic and "phaenogenetic" phenomena. If, as seems likely on general considerations, the effect is common to most organisms, it should be possible to produce, "to order," enough mutations to furnish respectable genetic maps, in their selected species, and, by the use of the mapped genes, to analyze the aberrant chromosome phenomena simultaneously obtained. Similarly, for the practical breeder, it is hoped that the method will ultimately prove useful. The time is not ripe to discuss here such possibilities with reference to the human species.

The writer takes pleasure in acknowledging his sincere appreciation of the cooperation of Dr. Dalton Richardson, Roentgenologist, of Austin, Texas, in the work of administering the X-ray treatments.

UNIVERSITY OF TEXAS

H. J. MULLER

SCIENTIFIC APPARATUS AND

LABORATORY METHODS

AN INSTRUMENT FOR REPEATED DETERMINATIONS OF BLOOD VISCOSITY

IN AN ANIMAL1

IN experiments where it is desirable to make repeated determinations of the viscosity of the blood of an animal, the withdrawal of the amount of blood

1 From the Physiological Laboratories of the University of Chicago and the University of Western Ontario, London, Canada.

insulated terminal "N" is in contact with the metal handle of the movable plate; this makes an electrical contact and the signal magnet records the time taken for the blood to be driven through the capillary tube under a known pressure. This time when compared with that required for water under the same pressure gives the relative viscosity of the blood.

It is evident that any number of determinations may be taken without decreasing the amount of blood in the animal. The electrical recording of the time is of advantage in reducing the error due to the reaction time of the experimenter. Because of the short time the blood for determinations remains in the

bulb, a bath for temperature control is not thought

necessary.

In order to test the accuracy of the instrument, a series of experiments was carried out in which the relative viscosity of 7 per cent. gum arabic was determined by means of this instrument and the same procedure carried out with the Oswald viscosity pipette, water being taken as unity; in thirty determinations with each instrument it was found that the relative viscosity of the gum solution when determined with this instrument was 3.76 while with the Oswald type it was 3.78. These results appear to be well within the range of experimental error. The determinations were made at room temperature and the pressure on the fluid maintained at 70 mm. Hg. The author is indebted to Professor A. J. Carlson for his helpful suggestions and criticisms of this work and to Mr. F. W. Claassens for his cooperation in construction of the instrument.

RUSSELL A. WAUD

VIABILITY

According to McDonald1 (1922) the trophozoites of Balantidium of the pig become spherical when the intestinal contents are cooled to room temperature. McDonald also states that they live at room temperature not longer than eight hours. Accordingly, a thermos bottle was used to carry the material to the laboratory from the packing plant. No appreciable rounding was noted when the organisms were examined at room temperature. Therefore, the content of a bottle obtained January 3, 1927, was allowed to cool. Active, apparently normal, trophozoites were found in a sample taken from this bottle the next morning and on every subsequent morning until January 14. The relative numbers did not appear to diminish for about seven days, but then fell off very rapidly. The temperature of the contents of the bottle was taken after fourteen hours, and found to be 20° C. On several other occasions the organisms lived at room temperature for four days and on one occasion for seven days. The viability of trophozoites is also indicated by the fact that water from the trucks in which the pigs were transported from the cars was found to contain them; they remained perfectly normal in appearance at room temperature for twenty-four hours. Feces passed at least two hours previously by ten different pigs were collected from the pens at the packing plant. Trophozoites were found in seven of the ten samples. The pigs had been long in transit from Ohio and the feces were well formed so that they had to be torn apart in water before the trophozoites were freed. The latter appeared perfectly normal and swam about actively.

SPECIAL ARTICLES

BALANTIDIA FROM PIGS AND GUINEA-PIGS: THEIR VIABILITY, CYST PRODUCTION AND CULTIVATION

THE following data concerning Balantidium occurring in the pig and guinea-pig are deemed of sufficient importance to warrant a report at this time. An abundance of material from the pig has been obtained from two packing plants within several squares of the laboratory, and Dr. W. R. Stokes, of the Baltimore City Health Department, has kindly furnished guinea-pigs for autopsy that died as a result of experimental work. Thus far of the twenty examined, 55 per cent. were infected with Balantidium. A colony of rhesus monkeys which also harbor Balantidium is maintained by Dr. Carl Hartman, of the Carnegie Institution. This article is a progress report on a problem of host-parasite relations which was suggested to me by Dr. R. W. Hegner.

INFECTIVITY OF TROPHOZOITES

It seems to be the general opinion that ingestion of cysts must occur to set up an infection. (Fantham, Stephens and Theobald,2 1916); but Hegner3 (1926) injected trophozoites from the pig into the stomach of the guinea-pig, and, when the animal was killed one hour later, active, apparently normal trophozoites were found in the stomach, small intestine and cecum. This experiment has been repeated with success.

1 McDonald, J. D. 1922. "On Balantidium coli' (Malmsten) and Balantidium suis (sp. nov.) with an Account of their Neuromotor Apparatus. Univ. Calif. Pub. Zool., 20: 243-300.

2 Fantham, H. B., Stephens, M. D., and Theobald, M. A. 1916. "The Animal Parasites of Man," 900 pp. New York.

3 Hegner, R. W. 1927. "Host-Parasite Relations between Man and His Intestinal Protozoa.'' The Century Co., New York. (In press.)

Trophozoites of the pig Balantidium, grown in culture, were injected into the stomach of a guinea-pig four weeks old. It died during the night, but after eighteen hours trophozoites, gorged with starch grains, were found in the esophagus and cecum. That these were not inhabitants of the intestine before the experiment began is indicated by the fact that the latter are always translucent and move slowly, whereas those from cultures are blackened from ingested starch and move actively. Later another guinea-pig from the same litter was given trophozoites by stomach tube direct from the pig. This animal also died during the night and trophozoites were found the next morning in the ileum, jejunum and cecum. They were of two kinds in the cecum; (1) those that parasitize the guinea-pig and (2) the starch-filled forms of the pig. When it is considered that trophozoites may live in feces for ten days at room temperature it is probable that they may serve as well as cysts in transmitting infection.

CYST PRODUCTION IN THE PIG

McDonald (1922) states that trophozoites were in various stages of encystment in the lower colon and rectum of the pig and all encysted in the formed feces. In Baltimore during February and March no cysts were found until about thirty pigs, all of which were infected with Balantidium, had been examined. They were not numerous; about three hours were required to pick out twenty specimens. Because of their large size, cysts of Balantidium can be distinguished with a binocular microscope. Material can be diluted in a Syracuse watch glass and examined much more rapidly than when slides are used. Cysts can be picked out with a micropipette and studied under the compound microscope. In this work, as well as by McDonald (1922), the cysts were found to be very resistant to fixing and staining. The nucleus and other structures are not revealed when they are treated with iodine eosine or even when Mallory's haematoxylin is run under the cover glass after fixation with Schaudinn's fluid. When bodies resembling cysts were finally found the material was treated in bulk with hot Schaudinn's and stained with iron haematoxylin (method of Kofoid and Swezy). They were then picked out and positively identified. Walker1 (1913) and others state that fecal diagnosis for Balantidium in man and monkeys is unsatisfactory, because for long periods no cysts are passed. Walker (1913) states, as does McDonald (1922),

that cysts are frequent in feces of pigs. Walker and others express the opinion that for this reason infection in man is usually contracted from pigs. Data obtained in Baltimore, however, indicate that cysts may be as scanty in pigs as in man, monkeys and guinea-pigs.

CULTIVATION

Much experimental work has been done recently and is being continued on cultivating parasitic protozoa. The first media tried by myself were made according to directions given by Dobell and Laidlaw5 (1926) for Endamoeba histolytica. On one occasion trophozoites lived seventy-two hours and multiplied. The medium consisted of an inspissated human serum slant plus a fluid of Ringer's solution without dextrose, nine parts, and human serum, one part. Sterile rice starch was added. Transplants from this culture failed. Various egg media were tried with negative results. Walker (1913) concluded, as a result of experiments, that a 0.5 per cent. NaCl solution is best suited for Balantidium. Barret and Yarbrough (1921) cultivated Balantidium coli successfully in a medium 0.5 per cent. NaCl, fifteen parts, plus human serum, one part. Their cultures were maintained thirty-two days. In order to ascertain whether Walker's data could be applied in the cultivation of Balantidium from the pig the concentration of the medium in which the organism lives was determined. Feces from the cecum were filtered through filter paper and the freezing point method employed, with a Beckmann thermometer. The reading of filtrate from fresh feces was minus 0.70° C.; of filtrate from feces kept forty-eight hours in the laboratory minus 0.79° C.; and of feces kept 168 hours it was minus 0.72° C. The freezing point of blood serum is about minus 0.6° C., and is isotonic with an 0.85 per cent. NaCl solution. Walker found this hypertonic for Balantidium, and preliminary trials carried out here seemed to confirm this conclusion. Hence it was thought that the hypertonicity of 0.85 per cent. NaCl is due to an excess of inorganic ions. Accordingly, 10 cc of each of the above fecal filtrates were evaporated to dryness; and the organic matter driven off by heating. The residue was taken up in 10 cc of distilled water and the freezing point found to be minus 0.17° C. This is isotonic with a NaCl solution of about 0.25 per cent. On the basis of the above data an attempt was made to prepare synthetic media,

5 Dobell, C., and Laidlaw, P. P. 1926. "On the Cultivation of Endamoeba histolytica and Some Other Entozoic Amoebae." Parasit., 18: 283-318.

6 Barret, H. P., and Yarbrough, N. 1921. "A Method for Cultivation of Balantidium coli,'' Amer. Journ. Trop. Med., 1: 161.