The chromatoid body of Entamoeba invadens

Experimental

604

BRIEF THE

CHROMATOID

BODY

D. C. BARKER Department

of Zoology,

Cell Research 15, 604-639 (1958)

NOTES OF ENTAMOEBA

INVADENS

and K. DEUTSCH University

of Edinburgh,

Scotland

Received August 4, 1958

CHROMATOID bodies

are found in the cysts of nearly all Entamoeba. They are rod or bar shaped cellular inclusions, up to 10 p long, which stain deeply with basic stains. Deutsch and Zaman, using the electron microscope, found that in Entamoeba invadens they consist of small particles (about 200 A in diameter) arranged in a crystalline pattern. There is some indication that these particles are composed of smaller units. Chromatoid bodies have also been observed in the trophozoite of Entamoeba invadens, where they are smaller and more numerous than in the cyst [l] and in Entamoeba histotyfica [2]. Ray and Sen Gupta [S] report that in Entamoeba histolytica the bodies contain varying amounts of DNA and RNA. The bodies have been regarded as excess chromatin thrown off during division [4], or as food stores [2, 51. According to Ray and Sen Gupta [8] they play a part in the deposition of the cyst wall. We have attempted a more comprehensive histochemical study of these interesting inclusions and an analysis of their changes in relation to the life cycle of the organism Material and Methods.-The organisms were cultured in Jones’ medium at 25°C [6]. Fixation was mainly carried out in Schaudinn’s fluid, but also Carnoy’s 1 and 2, Champy’s, Flemming’s, Zenker’s, Serra’s fixatives, osmium tetroxide, formalin and sublimate acetic acid were used [3, 91. Table I contains a list of the histochemical tests carried out. Trophozoites treated with ribonuclease in the living state (see Table I, B, col. 5) were studied also in the electron microscope. They were first mixed in osmium tetroxide, dehydrated, embedded in a methacrylate mixture according to standard procedure and sectioned at 250 A. For investigation of the cyclic changes of the bodies in the trophozoite, samples were taken in intervals starting from the third day after inoculation. In each sample the number of bodies larger than 0.5 p was counted and the length of the longest axis was determined. The cysts were studied in a similar way, but the whole culture was treated with distilled water for 24 hours to get rid of the trophozoites and to avoid the formation of new cysts. Results and Discussions.-The histochemical tests (see Table I) indicate that the bodies consist mainly of large amounts of ribonucleic acid and some unspecified proteins. We could not demonstrate the presence of desoxyribonucleic acid as in Entamoeba histolytica. Ribonuclease apparently did not affect the cyst (Table I, B, col. 5), as presumably the large enzyme molecules could not penetrate through the cyst wall. A preliminary electron microscope study shows that the globular particles which form the bodies, appear to be flattened after treatment with the enzyme (comExperimental

Cell Research 15

iron [ 1O]

[l] et al. [7]

De Bruyn

Wenrick 1251 Rafalko [23] Michaelis 1201 Kurnick [ 171

5. Nucleic acid tests. (a) Feulgen nuclear reaction. (b) Toluidine blue. (c) Methyl green pyronin.

Armstrong

Burdon [6] Ray and Sen Gupta [24]

4. Lipid tests. (a) Sudan black. (b) Neutral red.

6. Fluorescent microscopy. (a) Acridine orange. (b) Trypaflavin. (3,6-Diamino-10 methylacridinium chloride)

Glick [ll] Mazia el al. [19]

Hale [12] Bensley [2 J Hotchkiss 1151 McManus [18]

Dobell

Reference

3. Protein tests. (a) Berg’s ninhydrin. (b) Bromphenol blue.

2. Glycogen detection tests. (a) Bauer Feulgen. (b) Best’s carmine. (c) Periodic acid Schiff’s.

1. Basic stain. (a) Heidenhain’s haematoxylin.

Test

TABLE

stained

of chromatoid bodies

reacfion.

Orange

Bright

orange

Negative Negative Deeply stained Pyronin positive green negative

Negative Negative

methyl

Doubtfully positive Deeply stained

Negative Negative Stained membrane prevents definite statement but probably negative

Deeply

Reaction

I. A. Staining

as a peripheral with a central

ring of chronucleolus.

only very

poorly

in cytoplasm. in cytoplasm. stained.

lightly stained. stained.

yellowish

Nucleus

green.

green yellow.

Nucleus

Nucleus as in 1. Nucleus methyl green positive pyronin negative.

Nucleus

Fat droplets Fat droplets

Rest of the cytoplasm Rest of the cytoplasm

Glycogen mass in cysts and staining spots in trophozoites. Deeply stained membrane prevents visualisation of contents.

Nucleus matin

Casual observation

9 n” 3 si n” -. 5 a” e 2

r e <

a 2 Q B C(. R

2 cb g

Brachet Brachet

4. Ribonuclease digestion fixed material.

[4] [5]

[3)

Vendrely-Randaval

5. Ribonuclease on living material treated for 4 hours in a solution of 10 mg ribonuclease in 10 cc water.

hydrochloric

3. Hot normal acid.

[9] [25]

and Ris [22]

Di Stephano

Brachet

acid at 4’C.

2. 10 % perchloric

Pollister

Reference

of nucleic acids followed

on

acid.

I. B. Extraction

1. Hot trichloracetic

Test

TABLE

with toluidine

I-I,0 at 6O’C. Slight reduction in basophilia. Cold H,O and HCl. Bodies stained in the normal way. Results varied with the fixative used. Schaudinn’s practically ineffective, as was Zenker’s fixed material. Carnoy’s and Serra’s both showed big reduction in basophilia. No loss of basophilia

Unstained

Unstained after 4 hours’ treatment

Substantial loss in basophilia in trophozoite bodies but not in cyst. Changes in the microstructure (EM)

H,O.

stained.

H,O at 4°C. Bodies normally

Unstained.

with

in baso-

Slight reduction bodies. Stained normally. Stained normally.

Hot H,O. philia of Cold H,O. Cold TCA.

Controls (H,O = Distilled water)

blue or methyl green pyronin.

Unstained

Reaction of body after extraction

by staining

The chromatoid body of Entamoeba invadens

I’ig. I.-Chromatoid Fig. Z.-Chromatoid

body. Fixation body. Treated

osmium. with ribonuclease.

Fixation

607

osmium. Experimental

Cdl Research 15

608

D. C. Barker

pare Figs. 1 and 2). They consist sented in the electron microscope with electron transparent centres note that in formalin fixed, but tened (Fig. 4). TABLE II.

and K. Deufsch

now of linearly arranged subunits which are repreas electron transparent dots (diameter about 70 A) (diameter about 30 A) (Fig. 3). It is interesting to untreated cysts, the particles appear also to be flat-

Average number and size of bodies (larger than 0.5 ,u) in trophozoite and cyst.

Day 3 4 5 6 7 8 9 10 11 12

Trophozoite Number of Average bodies length per cell P 0.5 0.6 0.3 1.5 3.5 3.3 2.3 4.4 2.9 3.0

1.7 1.8 2.4 2.2 2.2 2.8 2.8 3.2 4.3 4.2

Hour

cyst Number of bodies per cell

4 16 24 48 72 96 120

1.4 2.1 1.7 2.1 3.7 4.4 3.3

Average

length P 4.8 4.9 4.2 4.3 3.0 2.3 2.1

Analysis of the changes of the bodies larger than 0.5 ,U in relation to the life cycle shows an increase of their number and their size with increasing age of the culture (Table II). The point of maximum size is reached in the late trophozoite and the early cyst. The number, however, drops during encystment. With increasing age of the cyst, the number increases again and the size decreases. Electronmicrographs show that the early trophozoite contains a great number of bodies smaller than 0.5 ,u, and it appears likely that the larger bodies are formed by aggregation of the smaller ones, and that the total number of bodies actually decreases in the trophozoite until encystment, as no. small bodies were detected in the early cyst [I]. The size and the chemical composition of the particles indicates that they could have a function similar to the RNA particles in other cells [7], but there is also a certain resemblance between the particles and viruses, and we shall try to clarify by further investigations the significance of these peculiar cellular inclusions. Summary.-Histochemical studies have shown that the chromatoid bodies consist mainly of ribonucleic acid and some unspecified proteins. A preliminary electron-microscope study has shown that the globular particles which form the bodies are flattened after treatment with ribonuclease. The average size of the bodies over 0.5 ,U increases with the age of the culture in the Trophozoite and decreases again in the cyst. Experimental Cell Research 15

The chromafoid body of Enfamoeba invadens

Fig. 3.-Chromatoid Fixation osmium. Fig. 4.-Chromatoid

body.

Treated

body. Fixation

with

ribonuclease

showing

fine structure

609

of the particles.

formalin. Experimental

Cell Research 15

Helen P. Sorokin

610

The authors wish to express their gratitude to Professor M. M. Swann for helpful discussions, and also to the Melville Trust for Cancer Research who equipped the electron microscopy laboratory in which part of the investigations were carried out. REFERENCES G. R., Exptl. Cell Research 11, 640 (1956). C. M., Stain Technol. 14, 47 (1939). J., Quart. J. Microscop. Sci. 94, 1 (1953). -Nature 175, 853 (1955). -Exptl. Cell Research 10, 195 (1956). BURDON, K. L., J. Bacferiof. 52, 665 (1946). DE BRUYN, R., FARR, H., BANKS, R. S. and MORTHLAND, F. W., Expfl. Cell Research 4, 174 (1953). DEUTSCH, K. and ZAMAN, V., Exptt. Cell Research. (In press.) Dr STEPHANO, H. S., Science 115, 316 (1952). DOBELL, C., The Intestinal Protozoa of Man. M.R.C.J. Bale, Sons, and Danielson, London 1921. GLICK, D., Techniques of Histo-Cyto-chemistry. Interscience Publ. Inc., New York, 1949 HALE, -A. J., Intern. Rev. Cyfol. VI, 193 (1957). HARTMANN, M., Arch. Profisfenk. 24, 163 (1912). HOARE, C. A., Handbook of Medical Protozoology. Tindal and Cox, London, 1949. HOTCHKISS, R. D., Arch. Biochem. 16, 131 (1948). JONES, W. R., Ann. Trop. Med. Parasif. 40, 130 (1946). KURNICK, N. B., Intern. Rev. Cyfol. IV, 221 (1955). MCMANUS, J. F. A., Nature 158, 202 (1946). MAZIA D., BREUER, P. A. and ALFERT, M., Biol. Bull. 104, 69 (1953). MICHAELIS, L., Cold Spring Harbor Symposia Quanf. Biol. 12, 13 (1947). PALADE, G. and SIEKEVITZ, P., Biochem. Cyfof. 2, Suppl. 85 (1956). POLLISTER, A. W. and RIS, R., Cold Spring Harbor Symposia Quanf. Biol. 12, 147 (1947) RAFALKO, J. S., Stain Technol. 21, 91 (1946). RAY, H. N. and SEN GUPTA, P. C., Buff. Calcutta School Trop. Med. 3, 1, 25 (1954). VENDRELY-RANDAVAL, C., Compt. Rend. Acad. Sci. 228, 607 (1949). WENRICK, J. P., J. Parasifof. 27, 1 (1941).

1. ARMSTRONG, 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

BENSLEY, BRACHET,

THE

MOTION

OF PLANT

HELEN

P. SOROKIN

Winchester, Received

T HE

SPHEROSOMES

Mass.,

September

U.S.A. 4, 19.58

motion of spherosomes may ideally be studied in epidermal cells from the lower side of rosette leaves in Campanula persicifolia L., because there they are large in size and few in number. In the studies of temperature effects on living cells Alexandrov [l] had found in this material the motion of spherosomes to be a sensitive criterion of cell vitality. Experimental

Cell Research 15