Zbl. Bakt. Hyg. A 264, 363-372 (1987)
Localization of Silver Deposits on Pneumocystis carinii Treated with Gomori's Methenamine Silver Nitrate Stain~· HISAO YOSHIKAWA and YUKIO YOSHIDA Dept. of Medical Zoology, Kyoto Prefectural University of Medicine, Kyoto 602, Japan
With 12 Figures' Received February 11, 1986 . Accepted April 25, 1986
Summary The question which portion of the pellicle of Pneumocystis carinii is actually stained by the Gomori's methenamine silver nitrate (GMS) method was solved with the aid of electron mIcroscopy. Silver particles were specifically deposited on the electron-lucent middle layer of the precyst, cyst and ruptured cyst, whereas such deposition was not found on the pellicle of the trophozoite, probably due to the absence of the electron-lucent middle layer in the trophozoite. These deposits increased in proportion to the duration of staining and to the thickness of the lucent middle layer. Therefore, the lighter colored organisms as observed under the light microscope are considered to be the precysts because they have thinner GMS-positive middle layers than those of cysts. So-called parentheses-like structures or rings are one of the most characteristic light microscopic features of P. carinii cysts when stained with GMS. The present study indicates that these structures must be of cyst wall origin rather than of cytoplasm origin, because the thickened part of the cyst wall showed a dense deposition of silver particles, probably corresponding to the parentheses-like structure. On the other hand we also observed, in the endogeny form of trophozoite, a thin GMSpositive layer in the parent pellicle though this pellicle showed no differences from those of daughter and common trophozoites when observed by means of ultrathin section microscopy. Zusammenfassung Mit Hilfe der Elektronenmikroskopie wurde die Frage gel6st, welcher Teil der Membran (pellicle) von Pneumocystis carinii durch Gomoris Methenamin-Silbernitrat (GMS)Methode tatsachlich angefarbt wird. " Presented at the 5th German-Japanese Cooperative Symposium on Protozoan Diseases, Tokyo, Japan, Sept. 25-28, 1985. Contribution No. 541 from the Department of Medical Zoology, Kyoto Prefectural University of Medicine. This work was supported by Grand-in-Aid for Scientific Research from the Ministry of Education, Science and Culture.
H. Yoshikawa and Y. Yoshida
Die Silberpartikeln schlugen sich speziell auf der elektronen-durchlassigen mittleren Schicht von Prazyste, Zyste und geplatzter Zyste nieder, wogegen sich auf der Membran des Trophozoiten ein solcher Niederschlagnicht fand, was vermutlich auf das Fehlen der elektronendurchlassigen Mittelschicht beim Trophozoiten zuriickzufiihren ist. Diese Ablagerungen nehmen proportional zur Dauer der Anfarbung und der Starke der durchlassigen Mittelschicht zu. Die unter dem Lichtmikroskop heller gefarbten Organismen werden daher als Prazysten angesehen, weil sie diinnere GMS-positive Mittelschichten aufweisen als die Zysten. Sogenannte klammerartige oder ringf6rmige Strukturen sind fiir mit GMS angefarbte P. carinii-Zysten unter dem Lichtmikroskop charakteristisch. Die vorliegende Untersuchung weist darauf hin, daB diese Strukturen eher aus der Zystenwand als aus dem Zytoplasma hervorgehen, da der verdickte Teil der Zystenwand eine dichte Ablagerung von Silberpartikeln aufwies, was wahrscheinlich der klammerartigen Struktur entspricht. Andererseits beobachteten wir in der Endogenieform des Trophozoiten auch eine diinne GMS-positive Schicht in der urspriinglichen Membran, obwohl sich diese elektronenmikroskopisch nicht von der Membran von Tochter- und gew6hnlichen Trophozoiten unterschied. Introduction Pneumocystis carinii is a causative organism of fatal pneumonia that is one of the most important opportunistic infections in patients who are receiving immunosuppressive therapy for their underlying diseases and conditions such as cancer, leukemia, malignant lymphoma, renal transplantation, collagen diseases etc. Recently, this type of pneumonia has been recognized as the most critical complication of acquired immune deficiency syndrome (AIDS). The methenamine silver nitrate stain was first used by Gomori (1946) to demonstrate glycogen and mucin. Grocott (1955) modified this technique to be used for the staining of fungi. Since then, Gomori's methenamine silver nitrate (GMS) stain has been known as reliable for the staining of P. carinii cysts (1, 3, 10, 12, 15, 19) and is now widely used for the diagnosis of P. carinii pneumonia (2, 5-7, 14, 16-18, 20,
22-26,30,31). The cyst wall and so-called parentheses-like structures or rings are specifically stained by this method (3, 12-14, 16, 18,20,24). However, the question whether the parentheses-like structure originates in the cyst wall or in the cytoplasm has remained unsolved (14,16,18,20,24,27). The present study attempts to make clear, with the aid of electron microscopy, which portion of the pellicle of P. carinii is actually stained by GMS. Materials and Methods Provocation of Pneumocystis carinii Seven-week-old Wistar male rats of approximately 200 g body weight received subcutaneous injections of 3.75 mg prednisolone acetate twice a week for several weeks. They were kept in a vinyl isolator and provided drinking water containing 1 mg/ml tetracycline to prevent bacterial infection (4). Procedure of fixation and Gomori's methenamine silver nitrate stain After 10 weeks of prednisolone treatment, the rats were perfused with 2.5% glutaraldehyde in 0.05 M cacodylate buffer via the right cardiac ventricle under sodium pentobarbi-
Silver Deposits on P. carinii Treated with GMS
tal anesthesia. The same fixative was introduced into the lungs through a tracheal cannula. The lungs were then removed en bloc and kept in the same fixative for 30 min at 4°C. After having been cut into small blocks, these were immersed in the same fresh fixative for 2 hat 4°C. They were divided into two groups, one for GMS staining and the other for control without staining. After the tissue blocks for GMS staining had been placed, for 1 h, into the same buffer with 8.2% sucrose, they were cut into 40 !-lm thick sections using a cryostat at -20°C. These sections were then stained with the working solution of GMS (9) for 30, 45, 60, 80 and 100 min, followed by post-fixation with 1% osmium tetroxide for 1 h at 4°C. For the control, the glutaraldehyde-fixed tissue blocks were post-fixed with 1% osmium tetroxide for 2 h at 4°C. Both materials were dehydrated with graded series of acetone and embedded in epoxy resin. Ultrathin sections obtained with the aid of Porter Blum MT-1_ ultramicrotome with glass knife were stained with utanyl acetate and lead citrate and examined with a JEM 1005 electron microscope.
Results The development of Pneumocystis carinii can generally be divided into three stages, trophozoite, precyst and cyst. At first the ultrastructures of P. carinii obtained from the control materials are shown as a reference for comparison with those stained with GMS (Fgis. 1-3). The trophozoite is characterized by having polymorphic external shape and 20-30 nm thick pellicle consisting of an electron-dense outer layer and an inner unit membrane (Fig. 1). The precyst is considered to be a transitional stage from trophozoite to cyst. The pellicle of the precyst increased in thickness from 40 to 90 nm, and an electron-lucent middle layer became apparent between the electron-dense outer layer and the inner unit membrane (Fig. 1). During the development from precyst to cyst, the pellicle reached a thickness of 100 nm or more. At this time, a partially thickened portion of the pellicle appeared showing a remarkable increase of the electron-lucent middle layer (Fig. 2). In the endogeny form of the trophozoite, the pellicle of the parent trophozoite did not show any difference in structure from that in daughter or common trophpzoites (Fig. 3). In the ultrastructures of P. carinii stained with GMS, the silver particles were specifically deposited on the electron-lucent middle layer of precysts, cysts and ruptured cysts, and such deposition was found neither on the electron-dense outer layer nor on the unit membrane except in the case of the longest duration (100 min) of staining (Figs. 4-11). These silver deposits increased in proportion to the increase in duration of staining and to the thickness of the lucent middle layer (Figs. 4-9). When the duration of staining was as short as 45 min, the silver particles were found to be slightly dispersing on the electron-lucent middle layers (Figs. 4, 5). Even in the electron-lucent middle layer which was recognized as a uniform layer in the control sections, it was noted after more than 60 min staining that the outer half of this layer was more densely impregnated with silver particles than the inner half facing the unit membrane (Figs. 6-9). When the duration of staining was unusually prolonged (e.g. 100 min), the silver deposition was recognized not only on the lucent middle layer but also on the dense outer layer, and sometimes on the outside of the pellicle (Fig. 9 Re). It is of interest to note that the silver deposition ws not found on the trophozoite pellicle (Fig. 9 T) even with such long duration of staining as 100 min. In cysts and ruptured cysts, the thickened portion of the pellicle showed a heavier deposition of silver particles than other parts of the pellicle (Figs. 6, 7, 10). On the
Silver Deposits on P. carinii Treated with GMS
other hand, the silver deposition was also seen in some cytoplasmic vacuoles of precysts (Figs. 4, 8) as well as in the pellicle of ruptured cysts phagocytized by neutrophils (Fig. 11). It is not infrequent to find the endogeny form of trophozoite (Fig. 3), although the position of this form in the life cycle of the organism is still unclear. When this form was stained with GMS for 80 min, the silver deposition was recognized on the inner part of the electron-dense layer of the parent pellicle (Fig. 12) although such deposition was not seen in the daughter pellicle. Discussion When P. carinii was stained with GMS and examined by the electron microscope, several grades of silver deposition were discriminated on the electron-lucent middle layer of the pellicle of precysts, cysts and ruptured cysts. However, no silver deposits were found on the pellicle of the trophozoite probably due to the absence of the lucent middle layer in this stage. Therefore, it is apparent that the portion which was stained in dark brown by GMS in light microscopy must be the lucent middle layer of the pellicle. 1n other words, GMS discloses only the stages of the development which have the lucent middle layer. As is commonly known in light microscopy, P. carinii stained with GMS will show various grades of brown color from light to dark. Among these, lighter colored organisms must be precysts and darker ones cysts and ruptured cysts. There was little silver deposition in the pellicles of the trophozoites even after strong staining as long as 100 min. Even in ruptured cysts phagocytized by neutrophils, the pellicle was strongly impregnated with silver particles. A single observation was reported on the ultrastructure of P. carinii stained with GMS (11). The authors fixed human autopsy material with formalin and osmium tetroxide, and showed some "single-walled organisms" whose pellicles were impregnated with silver particles. However, they showed neither a photograph nor gave a description of silver deposition on the cyst wall. We suppose that this silver deposition seen on some "single-walled organisms" was caused by the use of osmium tetroxide. Rambourg and Leblond (1967) mentioned that single use of osmium tetroxide fre-
..... Plate 1 Fig. 1. High magnification view of the pellicle of a trophozoite (T) and a precyst (P) without GMS stain. The pellicle of the trophozoite consists of an electron-dense outer layer (EDL) and an inner unit membrane (UM). In the precyst, an electron-lucent middle layer (ELL) appears between the outer dense layer and the inner unit membrane. x 50500 Fig. 2. A partially thickened portion of the lucent middle layer is seen in the pellicle of a precyst (arrowhead). x 14000 Fig. 3. An endogeny form of the trophozoite. Note that the pellicle of parent (PP) and of daughter (PD) trophozoites are similar in structure. x 12000 Fig. 4. A trophozoite (T) and a precyst (P) stained with GMS for 30 min. Note dispersed silver particles on the electron-lucent middle layer (ELL) of the pellicle of the precyst. x 26000
Silver Deposits on P. carinii Treated with GMS
quently caused nonspecific silver deposition. In general, glutaraldehyde is the sole suitable fixative for the following methenamine silver nitrate stain. A detailed examination of the electron-lucent middle layer in the present study revealed that the silver particles were more densely deposited on the outer portion of this layer than on the inner one which faced the unit membrane, though the lucent layer was recognized as a uniform image in the control section. This means that the outer part is much richer in GMS-affinitive substances than the inner part. In the pellicle of the precysts, several grades of silver deposition intensity could be distinguished. It seems that the substances stained with GMS are synthesized during the development of precysts which are the transitional stage from the trophozoite to the cyst. Furthermore, accumulation of these substances may be responsible for the formation of the electron-lucent middle layer of the pellicle. It is worthy to note that by strong GMS staining, the silver deposition was recognized also on the dense outer layer and sometimes even on the outer surface of the pellicle, but how this is brought about is difficult to explain at present. So-called parentheses-like structures or rings are one of the most characteristic light microscopic features of P. carinii cysts when stained with GMS. The question whether this structure originates in the cyst wall (3, 13, 16,20,27) or in the cytoplasm (14, 18, 24) has remained unsolved. The present study indicates that this structure must be of cyst wall origin because the thickened part of the cyst wall as well as of the ruptured cyst wall showed the heaviest deposition of silver particles, probably corresponding to the parentheses-like structure. We also observed the endogeny form of trophozoite which was first reported by Vossen et al. (1977, 1978). When this form was stained with GMS, the pellicle of the parent trophozoite showed a thin layer of deposited silver though in thin sections this pellicle was similar to that of common trophozoites. This means that the parent pellicle is somewhat different in its structural components from that of the common trophozoite. In other words, the parent trophozoite may synthesize to some extent substances similar to those found in the lucent middle layer of precyst and cyst.
~ Plate 2
Fig. 5. Two ruptured cysts (RC) stained with GMS for 45 min. Silver particles are also scattered on the lucent middle layers. x 13600
Fig. 6 and 7. Cysts stained with GMS for 60 min and 80 min respectively. Many silver particles are more densely deposited on the outer half of the lucent middle layer than the inner half of it. Note that the thickened portion of the pellicle (arrowhead) shows heavier deposition of silver particles than the other parts of it. x 16000 Fig. 8. Two different developmental stages of precysts (P) stained with GMS for 80 min. Note the different grades of silver deposition varying with the thickness of the lucent middle layer. Some silver reactions are also seen in the cytoplasmic vacuoles. X 9800 Fig. 9. A ruptured cyst (RC) and some small trophozoites (T) stained with GMS for 100 min. Strong silver deposition is seen not only on the lucent middle layer but also on the outer dense layer and sometimes on the outer surface of the pellicle (arrowheads), while few silver deposits are found on the pellicles of neighboured trophozoites. x 27000
H. Yoshikawa and Y. Yoshida
Plate 3 Fig. 10. A ruptured cyst stained with GMS for 80 min. Note that the thickened portion of the pellicle (arrowhead) shows the heaviest deposition of silver particles. x 16000 Fig. 11. Ruptured cysts (RC) and trophozoite (T) phagocytized by neutrophils. Only the pellicles of the ruptured cysts are stained with GMS. x 11000 Fig. 12. An endogeny form of trophozoite stained with GMS for 80 min. Note the thin silver deposition on the parent pellicle (arrowheads). x 29000
Silver Deposits on P. carinii Treated with GMS
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24. Ruskin, ]. and]. S. Remington: The compromised host and infection. l. Pneumocystis carinii pneumonia. ]. Amer. med. Ass. 202 (1967) 1070-1074 25. Singer, c., D. Armstrong, P. P. Rosen, and D. Schottenfeld: Pneumocystis carinii pneumonia: A cluster of eleven cases. Ann. lnt. Med. 82 (1975) 772-777 26. Singer, c., D. Armstrong, P. P. Rosen, P. D. Walzer, and B. Yu: Diffuse pulmonary infiltrates in immunosuppressed patients: Prospective study of 80 cases. Amer. J. Med. 66 (1979) 110-120 27. Vavra,]. and K. Kucera: Pne 1.tmocystis carinii Delanoe, its ultrastructure and ultrastructural affinities. J. Protozoo!. 17 (1970) 463-483 28. Vossen, M. E. M., P. ]. A. Bzckers, A. M. Stadhouders, A. M. G. Bergers, and]. H. E. Th. Meuwissen: New aspect~ of the life cycle of Pneumocystis carinii. Z. Parasitenk. 51 (1977) 213-217 29. Vossen, M. E. M. H., P.]. A. Beckers,]. H. E. Th. Meuwissen, and A. M. Stadhouders: Developmental biology of Pneumocystis carinii, an alternative view on the life cycle of the parasite. Z. Parasitenk. 55 (1978) 101-118 30. Walzer, P. D., D. P. Perl, D.]. Krogstad, P. G. Rawson, and M. G. Schultz: Pneumocystis carinii pneumonia in the United States: Epidemiologic, diagnostic, and clinical features. Ann. Int. Med. 80 (1974) 83-93 31. Wollschlager, c., F. Khan, ]. Kolavala, and V. Azueta: Pneumocystis carinii pneumonia in drug addicts. N. Y. State J. Med. 82 (1982) 352-354 Hisao Yoshikawa, Dept. of Medical Zoology, Kyoto Prefectural University of Medicine, Kawaramachi, Hirokoji, Kamikyo-ku, Kyoto 602, Japan