Methenamine Silver Nitrate Research Articles

Histology of retina overlying bacterial subretinal abscess and implications for treatment.

To evaluate the state of the retina overlying a subretinal abscess and its contents by histopathologic analysis and electron microscopy (EM) and to determine implications for treatment. Case report of a patient with a subretinal abscess secondary to Klebsiella pneumoniae infection who underwent pars plana vitrectomy, PPL, endolaser, retinectomy, abscess drainage, and retinal biopsy. The retinal biopsy was analyzed histologically using special stains, and EM of the abscess contents was performed. Postoperatively, the retina remained attached, and the patient regained visual acuity of 20/60. Culture of the vitrectomy specimen yielded K. pneumoniae. Retinal biopsy revealed a partially intact inner retina with destruction of the outer retinal layers in the setting of acute inflammation. Results of gram, Grocott-Gomori methenamine-silver nitrate, and acid-fast staining of the retinal biopsy specimen were negative. EM of the abscess contents revealed cellular debris with scattered inflammatory cells but no bacteria. This case demonstrates that aggressive surgical management of a subretinal abscess due to K. pneumoniae in an eye can result in useful vision. Management included retinectomy of the retina overlying the abscess that revealed photoreceptor destruction with intraretinal inflammation. Drainage of a subretinal abscess without removal of the overlying retina may limit treatment effectiveness and spare retina that is not functional.

Utility of reflex gomori methenamine silver staining forPneumocystis jirovecii on bronchoalveolar lavage cytologic specimens: A review

Pneumocystis jiroveci (Pj; formerly Pneumocystis carinii) is an opportunistic pathogen causing life-threatening pneumonia (Pneumocystis pneumonia) in immunosuppressed individuals. Its diagnosis is dependent on identification in bronchoalveolar lavage (BAL) specimens. Gomori's methenamine silver nitrate (GMS) stain has been advocated to highlight the organisms in BAL specimens. This study was performed to determine the utility of reflex GMS staining on all BAL specimens for identifying Pj.All BAL specimens from years 2000 to 2004 were processed as cytospins and stained with Papanicolaou (Pap) and GMS stains. A total of 2,984 BAL specimens were identified. A total of 116 (3.9% of total BAL) BAL specimens were diagnostic of Pj. The diagnostic specimens were grouped as follows: 103 (88.8% of total positive cases) Pj identified with both Pap and GMS staining; 11 (9.5% of total positive cases) Pj identified only with Pap staining; and 2 (1.7% of total positive cases) Pj identified only with GMS staining. In conclusion, the prevalence of Pj in BAL specimens is 3.9%, which can be attributed to improved management of immunocompromised patients. Performing reflex GMS staining on all BAL specimens does not improve the diagnostic identification of Pj since the majority (98.3%) of diagnoses can be rendered on Pap stained slides. A cost analysis for GMS staining on 2,879 GMS-negative BAL specimens was estimated at $143,950. Thus, from diagnostic and cost benefit perspectives, GMS staining can be recommended only on cases where Pap stain is negative, and the clinical presentation is consistent with Pneumocystis pneumonia.

Environmental stress and encystment of Pneumocystis carinii.

The structural elements and molecular events associated with encystment of Pneumocystis carinii are poorly understood. The cyst form of P. carinii is present in the lungs of infected hosts in low numbers as compared to the trophozoite; as few as 1/100 of the total organism number in severely infected animals are cysts [3]. It is not known if the cyst form is infectious or the degree to which it participates in eliciting immune responses from the host. Studies using drugs, that specifically prevent formation or destabilization of the cyst wall result in lower numbers of both trophozoites and cysts, suggesting that the cyst is an integral life cycle stage of P. carinii [1,4]. The environmental or organism signals that trigger encystment of P. carinii are unknown. Cysts appear late in the infection [3], suggesting that damage done to the host pulmonary system may create an inhospitable environment or reduce nutritional resources for the trophozoite, thereby triggering formation of the more environmental resistant and quiescent form, the cyst. This study sought to determine if a specific source of stress in the trophozoites’ environment is associated with encystment. MATERIALS AND METHODS Female Sprague Dawley rats (colony 202) were obtained from Harlan (Indianapolis, Ind.) and housed in an AAALAC-approved facility. Rats were immunosuppressed with dexamethasone (Dex) (0.36 mg/liter) in their drinking water for seven days. Then P. carinii organisms were introduced into rat lungs by transtracheal inoculation as previously described [2]. Broncheoalveolar lavage (BAL) fluid collection was performed as described previously [3]. Briefly, the trachea was exposed by blunt dissection. The thoracic cavity was opened by incisions through the ribcage and removing the ribcage to allow full expansion of the lungs during lavage. A 14-gauge Angiocath (Becton Dickinson, Infusion Therapy Systems, Sandy, UT) with a 10-mL syringe was inserted into the trachea, and the lungs were lavaged with aliquots of sterile, pyrogen-free saline solution at 378C, pH 7.3. The saline was recovered from the lung by withdrawing the plunger of the syringe in a deliberate manner. In some cases, only 5 mL of lavage fluid was instilled and recovered. Some lavage samples for pH determination were obtained from animals that were incubated at 48C for 24 hr after cardiac exsanguination. In these cases, lavage was performed as described above, with special attention to slow instillation and removal of lavage fluid to account for the increased friability of the tissue. Severity of P. carinii infection was determined by scoring numbers of organisms on histochemically stained impression smears of lung tissue using the semi-logarithmic scale of Barlett et al. [2]. The smears were stained with Wright’s-Giemsa stain for both trophozoite and cysts and with Grocott methenamine-silver nitrate stain for cyst forms [2]. For culture experiments, human embryonic lung (HEL 299) cells of fibroblast morphology (CCL-137; ATCC, Manassas, VA) were grown as monolayers on 24-well culture dishes using minimum essential medium (MEM) with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM nonessential amino acids, 10% fetal calf serum (FCS), 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Prior to inoculation of monolayers with P. carinii, the pH of the MEM was adjusted using 1 N HCl to 7.4, 7.3, 7.2, 7.1, 7.0, or 6.9. P. carinii samples were diluted to ten trophozoites per 10003 field to seed wells in the different pH media. Every other day, four individual wells were mixed using a Pasteur pipet, and 10-lL aliquots were

Histological and Histochemical Alterations in the Kidney Induced by Lead

Although lead intoxication is one of the most common forms of metal intoxication, the histochemical alterations in the renal tissues due to chronic lead exposure is limited and has not yet been well identified. A total of 60 male Wistar albino rats (Rattus norvegicus) were exposed to lead acetate trihydrate (0.0, 0.25, 0.5, 1.0 and 2% for 1 to 12 months) in drinking water to investigate the histological and histochemical alterations in the renal tissues due to lead. Chronic exposure to subtoxic doses of lead produced distinct progressive tubular, glomerular and interstitial damages. Tubular changes occurred earlier than glomerular and interstitial ones, and included anisokaryosis, nuclear pyknosis, karyomeglay, development of intranuclear and cytoplasmic inclusions together with tubular dilation, necrosis, vacuolization, tubular hyperplasia and solid tubular adenoma. The glomerular alterations were mainly mesangial hypercellularity, segmental proliferation, focal and segmental glomeruloscleriosis, glomerural hyalinization and glomerular tuft alterations. The findings indicate that lead produces significant histological and histochemical changes in the kidney that lead to severe complications.

Histological Detection of Aquatic Fungi by Uvitex 2B, a Fluorescent Dye

Conventional staining methods for fungal detection are time consuming, technically demanding, and instable dye-affinity in different fungal species, especially in aquatic fungi. In this study, Uvitex 2B [4, 4-BIS (2-di (2-hydroxyethyl) -amino-4 - (3-sulfophenylamino) -1, 3, 5-triazine-6-ylamino) -stilbene-2, 2'-disulfonic acid, sodium salt] was applied to detect fungal elements in paraffin sections of some aquatic animals. As a result, it was found that Uvitex 2B was superior to Gomori's methenamine-silver nitrate Grocott's variation, periodic acid-Schiff reaction and Schmorl's method for staining aquatic fungi in tissue sections. In addition, Uvitex 2B required much shorter time and less specialized skills in the staining procedure than Grocott. Although it has been known that oomycete fungi are difficult to be detected in histopahological sections because of their unstable stainabilities with other staining methods, Uvitex 2B provided excellent results to detect them in tissues of aquatic animals.

Cutaneous leishmaniasis: Texas case diagnosed by electron microscopy

A 78-year-old woman from south-central Texas without a history of travel outside the state presented with an erythematous papule on her right forearm. A biopsy specimen showed non-necrotizing granulomatous inflammation with negative stains for organisms. The lesion was recalcitrant to treatment with topical steroids, and a second biopsy specimen, obtained 3 months later, showed a histiocytic infiltrate in the upper dermis. Numerous, round, 1- to 30-μm bodies were seen within vacuoles in the histiocyte's cytoplasm. Giemsa, Grocott-Gomori methenamine-silver nitrate, and periodic acid-Schiff stains were negative. Electron microscopic examination of the paraffin-embedded tissue showed parasites diagnostic of leishmaniasis. On review of the literature, we found that 29 other cases of autochthonous cutaneous leishmaniasis have been reported in Texas. We emphasize the endemic nature of this condition in Texas. (J Am Acad Dermatol 2002;47:614-6.)

Estudo anatomopatológico e imunoistoquímico da pitiose em eqüinos naturalmente infectados

O trabalho teve como objetivo comparar a técnica de Grocott metanamina de prata (GMS) com o método imunoistoquímico de streptavidina-biotina marcada (LSAB) no diagnóstico da pitiose. Fragmentos de feridas cutâneas suspeitas de pitiose provenientes de 55 eqüinos foram processados pelas técnicas de hematoxilina/eosina, GMS e imunoperoxidase (LSAB). Trinta e quatro casos foram positivos pelo GMS, dos quais 28 apresentaram imunomarcação (LSAB) positiva para Pythium insidiosum. Os seis casos restantes apresentaram diagnóstico compatível de zigomicose. Foram diagnosticados como tecido de granulação com infiltração de eosinófilos sem áreas de necrose (nove casos), tecido de granulação com infiltrado de eosinófilos e presença de áreas de necrose (sete), habronemose (quatro) e sarcóide (um). Concluiu-se que a imunoperoxidase pelo método LSAB apresenta maior especificidade no diagnóstico de infecção pelo P. insidiosum do que pelo método GMS.

Diagnostic yield of bronchoalveolar lavage and bronchoscopic lung biopsy for detection of Pneumocystis carinii.

Objective To assess the need to perform a bronchoscopic lung biopsy (BLB) in addition to bronchoalveolar lavage (BAL) to obtain a definitive diagnosis of Pneumocystis carinii pneumonia. Design We retrospectively reviewed the results of concurrently collected paired BAL and BLB specimens to determine the diagnostic yield of both methods for the detection of P. carinii organisms. Material and Methods During a 3-year period, the BAL fluid specimens stained with a commercially available direct immunofluorescence monoclonal antibody (DFA) reagent and the BLB specimens stained with Grocott methenamine-silver nitrate (GMS) were assessed for the presence of P. carinii. BAL fluid was routinely collected from multiple sites and combined into a single specimen for testing. Results During the 3-year period of study, 119 patients were identified who had paired BAL fluid and BLB specimens tested for the presence of P. carinii. Of the 119 patients, 16 had either BAL fluid that could not be interpreted or BLB tissue that was inadequate. Of the other 103 patients, 21 had P. carinii pneumonia. The sensitivity of the DFA method on BAL fluid and of the GMS method on BLB was 95% and 43%, respectively. Conclusion For detection of P. carinii, the diagnostic yield is significantly higher for DFA-stained BAL specimens than for GMS-stained BLB specimens.

Diagnostic Yield of Bronchoalveolar Lavage and Bronchoscopic Lung Biopsy for Detection of Pneumocystis carinii

To assess the need to perform a bronchoscopic lung biopsy (BLB) in addition to bronchoalveolar lavage (BAL) to obtain a definitive diagnosis of Pneumocystis carinii pneumonia. We retrospectively reviewed the results of concurrently collected paired BAL and BLB specimens to determine the diagnostic yield of both methods for the detection of P. carinii organisms. During a 3-year period, the BAL fluid specimens stained with a commercially available direct immunofluorescence monoclonal antibody (DFA) reagent and the BLB specimens stained with Grocott methenamine-silver nitrate (GMS) were assessed for the presence of P. carinii. BAL fluid was routinely collected from multiple sites and combined into a single specimen for testing. During the 3-year period of study, 119 patients were identified who had paired BAL fluid and BLB specimens tested for the presence of P. carinii. Of the 119 patients, 16 had either BAL fluid that could not be interpreted or BLB tissue that was inadequate. Of the other 103 patients, 21 had P. carinii pneumonia. The sensitivity of the DFA method on BAL fluid and of the GMS method on BLB was 95% and 43%, respectively. For detection of P. carinii, the diagnostic yield is significantly higher for DFA-stained BAL specimens than for GMS-stained BLB specimens.

Aspergillus Endophthalmitis: An Unrecognized Endemic Disease in Orthotopic Liver Transplantation

Purpose: The authors discovered an unusually high incidence of Aspergillusendophthalmitis in an autopsy series of orthotopic liver transplantation recipients. This study was conducted to discern the frequency, topographic distribution, and potential significance of the infections.Methods: Autopsy reports from liver transplant patients were reviewed. All patients with Aspergillusendophthalmitis were studied by gross and histologic examination. Histologic sections were stained with Grocott-Gomori methenamine-silver nitrate and periodic acid-Schiff stains. Some Grocott-Gomori methenamine-silver nitrate stained sections were counterstained with hematoxylin-eosin. The distribution of ocular infections in the eye was determined for each patient. The organs infected were determined at autopsy.Results: The authors found seven patients with Aspergillusendophthalmitis. Six of these seven patients were from a group of 85 (7.1 %) orthotopic liver transplantation recipients. Fourteen (16.5%) orthotopic liver transplantation recipients had invasive pulmonary aspergillosis and ten (11.8%) had disseminated disease. The eyes were the second most common site of infection. Two patients had ocular involvement as the only nonpulmonary site of infection. Aspergillusendophthalmitis was diagnosed in only one patient before death. Infection was located posterior to the equator in all patients; three patients were anterior to the equator as well. The retina (5/7), vitreous (5/7), and choroid (3/7) were common sites of infection.Conclusions: This is the first report of Aspergillusendophthalmitis associated with orthotopic liver transplantation recipients. Patients with orthotopic liver transplants are unusually susceptible to invasive aspergillosis and Aspergillusendophthalmitis. Aspergillusinfection is frequently bilateral, begins posteriorly in the retina or choroid, and has vitreous involvement. Recognition of this entity is important because many patients die of disseminated Aspergillusinfection that may be detected early with bedside funduscopic examination.

Cutaneous Bacillary Angiomatosis in a Patient With Chronic Lymphocytic Leukemia

Bacillary angiomatosis is a recently described vascular disorder that is associated with infection by Bartonella henselae (formerly known as Rochalimaea henselae) and Bartonella quintana (formerly known as Rochalimaea quintana); this disorder usually occurs in patients with human immunodeficiency virus infection. We report a case of cutaneous bacillary angiomatosis that occurred in a patient with chronic lymphocytic leukemia. A 55-year-old man with chronic lymphocytic B-cell leukemia, Rai stage IV, presented with multiple angiomatous papules that clinically resembled pyogenic granulomas. Histopathologic examination revealed circumscribed lobules of small vessels with plump endothelial cells, numerous neutrophils, and abundant nuclear dust; these features were diagnostic for bacillary angiomatosis. The diagnosis was confirmed by the Grocott-Gomori methenamine-silver nitrate stain that revealed argyrophilic bacteria and by ultrastructural demonstration of bacillary structures with trilaminar walls. Treatment with clarithromycin led to complete resolution of the lesions within 4 weeks. This case emphasizes that (1) bacillary angiomatosis must be considered in the differential diagnosis of vascular lesions in immunocompromised patients without human immunodeficiency virus infection, (2) Grocott-Gomori methenamine-silver nitrate stain is a simple and satisfactory alternative to the Warthin-Starry stain for the demonstration of bacilli in this condition, and (3) clarithromycin is an effective oral antibiotic for the treatment of this disease.

Adiaspiromycosis

Adiaspiromycosis (ad"i-ah-spi"ro-mi-kósis) is a worldwide, noninfectious, nonarthropod transmitted fungal infection of lower vertebrates, most commonly rodents. Humans become an accidental host by inhaling dust-borne spores (conidia) of the saprophytic soil fungus, Emmonsia crescens (recently renamed Chrysosporium parvum variety crescens). We report 11 cases of this unusual deep mycosis from South America, Europe, and the United States. The severity of the disease depends on the number of spores inhaled. In limited inoculum, the disease remains localized (two cases), whereas in heavy inocula the fungus involves both lungs (nine cases) and presents as a diffuse reticulonodular infiltrate. In this disseminated form, patients usually complain of cough, dyspnea on exertion, and low-grade fever mimicking other systemic fungal infections and tuberculosis. It is difficult to unmask the fungus because it is not easily cultured nor is there a reliable serologic test. Therefore, a biopsy is required and the pathologist must recognize the large (ranging in size from 50 to 500 microns), round, Gomori methenamine-silver nitrate and periodic acid-Schiff reagent-positive spherules with a trilaminar wall. The spherules can be surrounded by either suppuration, epithelioid granulomas with or without necrosis, or concentric, hyalinized fibrosis. In the latter chronic stage, the organism may collapse, forming a variety of sizes and shapes thereby resembling other fungi, helminths, mineral particles, or inhaled pollen grains. Clinically, the infection most commonly regresses spontaneously, but may persist, or rarely progress, requiring surgical intervention with limited resection to attain cure.

One Minute Oxidation with Silver Impregnation for Fungi andPneumocystis carinii

AbstractTwo improved silver methods for fungi and Pneumocystis carinii that use a modified oxidizing agent are presented. The sections were first oxidized with a solution consisting of chromic, sulfuric, and glacial acetic acids for 1 min in the 60°C paraffin oven, thoroughly washed in running tapwater, and treated with sodium metabisulfite before they were impregnated in modified silver methenamine or ammoniacal silver nitrate solution. The sections were treated with sodium thiosulfate before and after gold chloride. They were counterstained with light green.This modified oxidation prevented staining of elastic and collagen fibers. The entire procedure was performed in approximately 40 min or less, depending on the temperature used in the silver impregnation. All fungi tested and P. carinii were stained in the same amount of time in both silver nitrate and gold chloride. (The J Histotechnol 15:307, 1992)

Pneumocystis carinii pneumonia in AIDS patients: clinical course in relation to the parasite number found in routine specimens obtained by fiberoptic bronchoscopy.

The aim of this study was to evaluate whether the amount of Pneumocystis carinii organisms found at fiberoptic bronchoscopy (FB) performed on HIV-positive patients correlated to the character of the P. carinii pneumonia (PCP). A consecutive series of 105 patients presented with 131 episodes of pulmonary symptoms requiring FB, and in 75 of these episodes a diagnosis of PCP was made. Specimens were stained with Giemsa and methenamine silver nitrate and the number of parasites found was given as: numerous, many, few or none. The following signs and symptoms were registered: cough, dyspnoea, fever, loss of weight, chest radiograph, haemoglobin, WBC, CD4 cell count, PO2 and HIV p24 antigen. The PCP was characterized by the clinical course: mild, moderate, severe, and by the outcome: pulmonary healthy, pulmonary insufficiency and death. No correlations between the number of P. carinii organisms and the clinical course or outcome of the PCP, the symptoms before the FB or the paraclinical examinations were found. In conclusion, the routinely obtained quantitative results of the microbiological examinations of material from the lungs were not correlated to the severity of the PCP.

Examination of Induced Sputum In the Diagnosis of Pneumocystis Carinii Pneumonia

The results of the examination of sputum induced by the inhalation of nebulized hypertonic saline in the diagnosis of Pneumocystis carinii pneumonia (PCP) are presented. In suspected cases of PCP in patients who were either HIV antibody positive or were receiving immunosuppressive therapy, 46 induced sputum specimens were stained using both Grocott's modified Gomori methenamine silver nitrate (GMS) and immunofluorescence staining. In 12 specimens P. carinii cysts were detected by both methods, in four specimens by GMS staining only and in five specimens by immunofluorescence only. The sensitivity of induced sputum examination in the detection of P. carinii cysts was increased by using both of these staining methods on each sputum specimen and the need for more invasive methods of diagnosis was reduced.

Improved detection ofPneumocystis carinii by an immunofluorescence technique using monoclonal antibodies

To assess whether a recently developed indirect immunofluorescent stain using monoclonal antibodies was more sensitive in detecting Pneumocystis carinii than the combination of Giemsa and methenamine silver nitrate stains which has routinely been used in the laboratory, 88 lavage fluid specimens and 34 induced sputum specimens were examined. All specimens were stained by five techniques: immunofluorescence using a combination of three monoclonal antibodies (from the National Institutes of Health, USA), immunofluorescence using a single monoclonal antibody (from Dakopatts), Giemsa, methenamine silver nitrate and toluidine blue O. Immunofluorescence using the monoclonal antibodies from the NIH was significantly more sensitive than any other single staining method and than the combination of Giemsa and methenamine silver nitrate staining. The study also showed that the cytospin centrifuge was very suitable for the preparation of slides with lavage fluid and processed induced sputum. Finally, the sensitivity of examination of induced sputum to detect Pneumocystis carinii was found to be 50% when compared with bronchoalveolar lavage fluid. However, this sensitivity may increase through practice.

Pneumocystis carinii Infection in Corticosteroid-Treated Cats

Corticosteroids were administered to produce Pneumocystis carinii infection in cats. Six of 10 cats, injected intramuscularly for 97-141 days with 2 mg/cat twice weekly of betamethasone sodium phosphate, developed a light infection with P. carinii. Six of 7 cats, injected intramuscularly for 11-168 days with 10-25 mg/cat weekly of prednisolone acetate, also developed a light infection with P. carinii. There was no significant difference in the infection rate between the sexes and ages of the cats. Using Giemsa staining and Gomori's methenamine silver nitrate stain, P. carinii organisms were indistinguishable morphologically from human and rat P. carinii. The cysts and trophozoites were usually present singly or in small groups, and they always were adhering to the periphery of alveoli. The inflammatory changes were inconspicuous except for the fact that alveolar macrophages often were seen. Corticosteroid-treated cats should be useful in the study of experimental P. carinii infection. This is the first reported case of experimentally induced P. carinii infection in cats.

Diagnosis of Pneumocystis carinii in sputum liquefied with the mucolytic agent dithiothreitol (sputasol).

1. Light microscopic investigations : Sputasol treatment did not affect the staining properties of either the cyst wall or the parentheses-like structures by Gomori's methenamine silver nitrate (GMS) stain. Detection of Pneumocystis carinii was easier in the Sputasol-treated preparations because it was possible to prepare thin smears of the sediment, and because the cysts were concentrated. The amount of time needed for microscopic examination was reduced to the decreased amount of sediment in the Sputasol-treated samples. 2. Transmission electron microscope investigations : In the ultrastructure of P. carinii cysts stained with GMS stain, the heavier deposition of silver particles on the internally thickened parts of the cyst wall corresponded exactly with the distinctive appearance of the parentheses-like structures seen in light microscopic observations.

A Simple and Highly Reproducible Staining Method for Fungi and Other Polysaccharide-Rich Microorganisms in Animal Tissues

A newly devised, simple and highly reproducible method for fungal staining is reported. Grocott's method, in which methenamine-silver nitrate solution is employed, has been widely used for the staining of fungi in tissue sections, but it frequently produces heavy background staining because of sudden and progressive reaction in the methenamine-silver nitrate solution. We therefore replace the latter solution with an ammoniacal silver nitrate solution. This new method yields more consistent results in fungal staining without background staining, since the reaction time in the ammoniacal silver nitrate solution is prolonged. The present method is considered superior to Grocott's method with regard to its simplicity and reproducibility.

Localization of silver deposits on Pneumocystis carinii treated with Gomori's methenamine silver nitrate stain

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 GMS-positive 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.