DIFFERENTIAL NUCLEAR COLORATION IN CILIATES 75 1

10. McKanna, J. A. 1973. Cyclic membrane flow in the inges- tive-digestive system of peritrich protozoans. I. Vesicular fusion at the cytopharynx. J . Cell Sci. 13, 663-70.

11. Mollenhauer, H. H. 1964. Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111-4.

12. Muller, M. 1972. Secretion of acid hydrolases and its intra- cellular source in Tetrahymena pyriformis. J . Cell Biol. 52, 478-87.

13. Nozawa, Y. & Thompson, G. A., Jr. 1971. Studies of mem- brane formation in Tetrahymena pyriformis. 11. Isolation and lipid analysis of cell fractions. J . Cell Biol. 49, 712-21.

14. ~ &- 1971. Studies of membrane formation in Tetrahymena pyriformis. 111. Lipid incorporation into various cellular membranes of logarithmic phase cultures. J . Cell Biol. 49,

15. - & - 1972. Studies of membrane formation in Tetrahymena pyriformis. V. Lipid Incorporation into various cel- lular membranes of stationary phase cells, starving cells, and cells treated with metabolic inhibitors. Biochem. Biophys. Acta 282,

16. Ricketts, T. R. 1971. Periodicity of endocytosis in Tetra- hymena pyriformis. Protoplasma 73, 387-96.

17. - 197 1. Endocytosis in Tetrahymena pyriformis. The selectivity of uptake of particles and the adaptive increase in cellu- lar acid phosphatase activity. Exp. Cell. Res. 66, 49-58.

J. PROTOZOOL. 21(5), 751-754 (1974).

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18. Rouser, G., Siakotos, A. N. & Fleischer, S. 1966. Quantita- tive analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids 1, 85-6.

19. Stossel, T. P., Mason, R. J. & Vaughan, M. 1972. Quanti- tative studies of phagocytosis by polymorphonuclear leukocytes. Use of emulsions to measure the initial rate of phagocytosis. J . Clin. Invest. 51, 615-22.

20. Subbaiah, P. V., & Thompson, G. A., Jr. 1974. Studies of membrane formation in Tetrahymena pyriformis. The biosynthesis of proteins and their assembly into membranes of growing cells. J . Biol. Chem. 249, 1302-10.

21. Thompson, G. A,, Jr., Bambery, R. J. & Nozaw-, Y. 1971. Further studies of the lipid composition and biochemical properties of Tetrahymena pyriformic membrane systems. Biochemistry 10,

22. -, - & - 1972. Environmentally produced alterations of the terahymanol : phospholipid ratio in Tetrahymena pyriformis membranes. Biochim. Biophys. Acta 260, 630-38.

23. Ulsamer, A. G., Smith, F. R. & Korn, E. D. 1969. Lipids of Acanthamoeba castellanii. Composition and effects of phagocytosis on incorporation of radioactive precursors. 1. Cell Biol. 43, 105-14.

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4441-46.

Differential Nuclear Coloration in Ciliates PILAR ALONSO*

Departamento de Protozoologia, Instituto "Jaime Ferrdn" de Microbiologia, C.S.I.C., Madrid-6, Espaiia

SYNOPSIS. Application of the triple stain of Grosso (methyl green-pyronin-orange G ) to the exconjugants of different spe- cies of ciliates produces a differential coloration of their nuclei. Both the old macronucleus or its remnants and the micro- nuclei bind methyl green whereas the newly developing macronucleus (macronuclear adage) has a selective affinity for the orange G present in the mixture. This finding holds true not only for hypotrichous ciliates, whose macronuclear anlagen are involved in the formation of polytene chromosomes, but also for other ciliates not included in the order Hypotrichida. Such a reaction of the new macronuclei apparently results from the presence of non-histone (acidic) proteins associated with the nucleic acids.

The staining method is fully described to provide a more general application.

Index Key Words: Euplotes eurystomus; Paramecium caudatum; Paramecium bursaria; Stentor roeseli; macronuclear an- lagen; cytochemistry ; light microscopy.

INCE the reintroduction of Pappenheim-Unna's technique S by Brachet (1 1) a number of papers have appeared using methyl green-pyronin staining for the identification of nucleic acids (compilation in ref. 18). Pappenheim ( 1 7 ) described another technic developed by Grosso using a mixture of the above mentioned basic dyes and orange G [quoted by Romeis (24)]. The latter method, in contrast to the former, has been rarely used, and it has even been pointed out (18) that for pure histochemical work orange G should be omitted.

In a previous paper ( 2 ) , however, using the mixture of Grosso on exconjugants of 2 hypotrichous ciliates, differential nuclear staining was obtained. The micronuclei and fragments of the old macronuclei showed marked affinity for methyl green, whereas the newly developing macronucleus had a selective affinity for orange G. Concomitantly, the presence of some special proteins were detectcd in the new organelle.

In a recent communication ( 3 ) , applying the triple stain of Grosso to other species of ciliates, we reported a differential coloration of the nuclear apparatus in the exconjugants of all the species studied. This result prompted us to describe here the method, including the modifications, with the hope that this technique will gain wider general use.

* The author is grateful to Prof. Jean Brachet for kindly improv- ing the manuscript.

MATERIALS AND METHODS

The following ciliates from their original sources were cultured in our laboratory, prior to experimentation, for periods ranging from a few months to several years: ( a ) Euplotes eurystomus isolated from a pond near Gudillos (Segovia) , and fed on small ciliates added to soil solution; ( b ) Paramecium caudatum isolated from a creek at El Berrueco (Madrid), and grown on lettuce infusion bacterized with Aerobacter aerogenes; (c) Para- mecium bursaria collected in the neighborhood of Madrid and maintained in diluted lettuce infusion; ( d ) Stentor roeseli isolated from a sample of stagnant water collected near Madrid, and cultured in soil solution with suspensions of Saccharomyces pastorianus added as food.

Whole exconjugants were fixed on albuminized slides with methanol-acetic acid (3 : l ) for 5 min at room temperature, and postfixed with 95% ethanol for 1 min. Staining was performed with methyl green-pyronin-orange G mixture for 5-8 min. After thoroughly draining the slides of the dye, the cells were differentiated in absolute ethanol holding each slide at an angle of - 45" from the horizontal and pouring the alcohol care- fully with the aid of a pipet over the upper end of the prepara- tion. The slides then were immersed in absolute ethanol for a few min ( 2 changes), cleared in xylol and mounted in Cedax. The unbuffered stain solution was used as originally prepared

752 DIFFERENTIAL NUCLEAR COLORATION IN CILIATES

Figs. 14. Ciliate preparations stained with methyl green-pyronin-orange G. 1. Portion of an early exconjugant of Euplotes eurystonus. Note the rounded macronuclear anlage at a central position (orange-yellowish) and 4 fragments of the old macro- nucleus (green), 2 of them rounded and 2 elongated. 2. Portion of an early exconjugant of Paramecium caudatum. A clear distinction is observable between 2 macronuclear anlagen (orange), numerous macronuclear fragments (green) and cytoplasm (pink). 3. Portion of an early exconjugant of Paramecium bursaria. At a central position 2 small macronuclear anlagen (yellow) (of which the one on the right is slightly out of focus) are visible. At the to are 2 micronuclei (green) while a t the bottom, the old macro- nucleus (green) can be seen. I n addition, there are a number of Cfiorella, a common symbiont of this species. 4. An exconjugant of Stentor roeseli. Three macronuclear anlagen (orange) and 2 macronuclear fragments (green) can easily be distinguished.

DIFFERENTIAL NUCLEAR COLORATION IN CILIATES 753

(24). T o 75 ml of distilled water was added, in sequence, the following saturated aqueous solutions : 3.2 ml methyl green (chloroform extraction of this stain was not performed), 6 ml pyronin and 1 ml orange G. The mixture was then allowed to stand overnight and filtered. Such a solution keeps well at room temperature for several months.

The nucleic acids (DNA and RNA) were also investigated by the methods of Feulgen and Brachet.

For demonstration of basic proteins the methods of Alfert and Geschwind ( 1 ) and Black and Ansley (10) were followed. Protein dye binding capacity was also tested with fast green at pH 2.2 (1) and mercuric bromophenol blue (15, 23).

Some slides were pretreated with 0.01% (w/v) pancreatic trypsin (Sigma, Type 111, recrystallized) in 0 . 5 ~ phosphate buffer at p H 6 for 15 min at 37C while others were exposed to 0.01% (w/v) RNase (Sigma) in distilled water for 30 min at the same temperature.

RESULTS As is well known, the process of conjugation in ciliates

generally results in the breakdown and resorption of the old macronucleus and the formation of a new one from a product of the synkaryon divisions. Thus, in a given exconjugant, the nuclear apparatus consists of a developing macronucleus or macronuclear anlage (the number of macronuclear anlagen varies with the species), a variable number of micronuclei and the old macronucleus or its remnants.

After the triple staining procedure, the high affinity of the macronuclear anlagen for the orange G of the mixture is striking, while the micronuclei and the fragments of the old macronucleus bind methyl green. The yellowish-orange color of the new macronucleus can be observed from the very begin- ning of macronuclear development. However, it fades away soon after this organelle has reached its maximal size in late exconjugants; from then on, its affinity for methyl green even- tually increases until the vegetative macronucleus is recon- stituted.

Some examples of the results obtained with Grosso’s technique are illustrated below. In early exconjugants of Euplotes eurystomus (Fig. 1 ) , one can distinguish the orange G-stained macronuclear anlage at a central position along with four fragments of the old macronucleus that stain with methyl green. Early exconjugants of Paramecium caudatum develop 4 macronuclear anlagen, only 2 of which are visible in Fig. 2; in addition, a number of macronuclear remnants resulting from the breakdown of the old macronucleus can be observed. In Paramecium bursaria, early exconjugants (Fig. 3 ) contain 2 developing macronuclei (yellow), two micronuclei (green) and the old macronucleus (green), besides a number of zoo- chlorellae, typical symbionts of this species. Finally, in Stentor roeseli exconjugants, a clear distinction between the orange- stained macronuclear anlagen and the green-stained macro- nuclear fragments can be discerned (Fig. 4 ) .

During the life cycle of the ciliates studied, no other nuclear element is stained by the orange G of the triple stain. I t must be pointed out, however, that the affinity of the macronuclear anlage for this acidic dye is not restricted to this organelle; the ciliary structures, in addition to the autophagic vacuoles (13) , also stained yellow.

Additional cytochrmical reactions have been performed and the results arr summarized in Table 1. The macronuclear anlage stains weakly after the Feulgen and Brachet methods; it also behavrs similarly with alkaline fast green and ammonia- cal silver, spccific staining procedures for histones, whereas the

TABLE 1 . Comparative histochemical reactions of nucleic acids and proteins in exconjugants of different ciliates.*

Macronuclear frag- ments or old

Treatment Macronuclear anlage (n ) macronucleus

Feulgen + + + + + Methyl green-

Methyl green-

Methyl green-

pyronin + green or + + + + green or bluish-green bluish-green

pyronin-orange G + + + yellowish- + + + + green orange

pyronin-orange G after trypsin + bluish green - - -

phenol blue + + + + blue + + blue

phenol blue after RNase

Mercuric brom-

Mercuric brom-

+ + + + blue + + + blue

Acid fast green + + + green + green

Alkaline fast green + green + + + green

Amrnoniacal silver + yellowish + + + + brown * + + + + : very intense; + + + : intense; + + : me-

dium; + : weak; - - - : none observed.

old macronucleus or its remnants react strongly under the same conditions. In contrast, fast green at p H 2.2 and mercuric bromophenol blue at low p H reveal an intense staining re- action in the macronuclear anlage. RNase digestion prior to treatment with mercuric bromophenol blue, has no effect on the staining of the macronuclear anlage.

After trypsin digestion, the mixture of Grosso stains the developing macronucleus a pale bluish-green. Thus, the ma- terial previously stained with orange G has been removed by the proteolytic enzyme.

DISCUSSION

In considering these results, we observe that the acidic dye, orange G, of Grosso’s mixture reveals a substance not detect- able by the standard methyl green-pyronin technique. In fact, the cytochemical data obtained indicate the presence of non- histone (acidic) proteins in the macronuclear anlage of dif- ferent ciliates.

On the other hand, polytene chromosomes, similar to the giant chromosomes of dipteran salivary gland cells, have been observed in the macronuclear anlage of several hypotrichous ciliates (4-8, 19, 21, 25), and the appearance of puffs in the polytene chromosomes of 2 different species of Stylonychia and other oxytrichidae has also been reported (5, 6, 20, 14). More- over the accumulation of acidic proteins in the polytene chromosomes of dipteran larvae seems to be one of the first events occurring upon gene activation ( 12, 26, 9 ) .

Among the species here studied, only Euplotes eurystomus exhibits typical polytene chromosomes (6, 22). The presence of such chromosomes has never been demonstrated in the macronuclear anlagen of Stentor or Paramecium; however, a “spirema” has long ago been reported in the developing macronucleus of the former (16) , and tiny filaments have re- cently been observed in the macronuclear anlagen of Para- mecium (3) . I t thus remains to be established at present whether the appearance of acidic proteins in the macronuclear

754 DIFFERENTIAL NUCLEAR COLORATION IN CILIATES

anlagen of ciliates is correlated with the building up of polytene chromosomes or is independent of such a process. It should be stressed in this connection, however, that a significant rc- organization must take place in an exconjugant before a normal vegetative individual is reconstituted.

We conclude that the triple stain of Grosso provides a dif- ferential coloration of the nuclei within a single cell. The markedly different colors may result from variations in proteins associated with nucleic acids and are reflected in orange G binding.

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13. Gil, R., PCrez-Silva, J. & Alonso, P. 1969. Fine structure of exconjugants of Stylonychia mytilus Ehrenberg. Progress Proto- zool. ( I I I rd Intern. Congr. Protozool., Leningrad), 61.

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20. -, - , Gil, R. & Jareiio, M. A. 1969. Puffing in the polytene chromosomes of Stylonychia mytilus Ehrenberg, in Strelkov, A. A., Sukhanova, K. M. & Raikov, I. B., eds., Progress in Protozoology, Proc. 3rd Int. Congr. Protozool., Leningrad, 1969, Nauka, Leningrad, 36.

21. Rao, M. V. N. 1966. Conjugation in Kahlia sp. with special reference to meiosis and endomitosis. 1. Protozool. 13, 565-73.

22. __ & Ammermann, D. 1970. Polytene chromosomes and nucleic acid metabolism during macronuclear development in Euplotes. Chromosoma 29, 246-54.

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24. Romeis, B. 1928. Guia-formulario d e Ttcnica Histolbgica (Spanish translation from the 1st German Edition), in Labor, S. A., ed., Barcelona.

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CORRECTION to Mundim et al. 1974. Simple nutrition of Crithidia deanei, a reduviid trypanosomatid with an endosymbiont. I . Protozool. 21(4), 518-21. On page 520, left column, under Antibiotics, line 8 should read: “of Table 1. The antibiotics used were: penicillin 25- 100,000.”