Mayer's Mucicarmine Research Articles

Sclerosing Central Mucoepidermoid Carcinoma: Rare Case Series and Review

Introduction:
 The most frequent malignant tumour of the salivary glands is mucoepidermoid carcinoma which accounts for 30% of all malignancies of the salivary glands. There are numerous histologic variations with various levels of differentiation. Sclerosing MEC (SMEC) is a rare subtype of MEC that has the potential to be misdiagnosed as a low-grade non-SMEC malignancy or a benign reactive condition. There have been no cases of the extremely rare sclerosing variant of central or intraosseous MEC reported as of yet.
 Case reports:
 We present two cases of Sclerosing Central MEC, in which histologic analysis revealed relatively well-circumscribed, nonencapsulated tumours made up of dispersed epithelial islands of low-grade MEC and extensive central sclerosis. Similar sclerotic stroma was present in the tumor in the second case, but the epithelial component was of intermediate grade. Both cases showed abundant intracytoplasmic mucin and positive Mayer mucicarmine and PAS stains.
 Conclusions:
 Sclerosing central mucoepidermoid carcinoma was identified as the cause. Both cases underwent a complete resection of the tumor and have since been free of the disease.

Nasal Cryptococcosis Caused by Cryptococcus gattii in Sheep in Bahia, Northeastern Brazil

Background: Cryptococcosis is a potentially fatal systemic mycosis that can affect several animal species. The disease is caused by the yeast species Cryptococcus neoformans and C. gattii and is usually associated with immunosuppressive diseases or debilitating factors. In Brazil, cryptococcosis in ruminants has been described in several states, but there have been only two instances of infection in sheep. Until the present, cryptococcosis caused by C. gattii has only been reported in goats. The objective of this study was to describe, for the first time, a case of nasal cryptococcosis caused by C. gattiiin a sheep in the state of Bahia.Case: The disease affected a 4-year-old Santa Inês sheep, created in extensive system in the municipality of Ribeira do Pombal, Bahia, Brazil. The animal presented weight loss, with an asymmetric volume increase in the right nasal cavity, accompanied by dyspnea and bloody nasal discharge. Samples of the tumor mass were collected for cytological analysis of yeast structures compatible with Cryptococcus spp. After cytological diagnosis, the sheep was sent to the Livestock Development Center of the Federal University of Bahia. On physical examination, there was a large and firm irregular mass, strongly adhering to the right nasal cavity and causing facial asymmetry. Surgical removal was attempted, but a marked infiltrative character of the lesion was observed, with extensive tissue destruction. Owing to the unfavorable prognosis, euthanasia was carried out followed by necropsy, in which a large irregular mass was observed that almost completely obliterated the right nasal cavity. Resorption of the right nasal bone and the dorsal and middle nasal turbinates was also observed. Fragments of the nasal lesion were collected, fixed in 10% formalin, routinely processed for histopathology, and stained with hematoxylin and eosin (HE) and Mayer's mucicarmine. Samples of this lesion were sent for molecular characterization of the etiologic agent by polymerase chain reaction (PCR). The histopathological evaluation showed hundreds of generally colorless rounded yeasts cells composed of a clear halo, surrounding a slightly basophilic structure, giving the tissue a vacuolated aspect, known as a “soap bubble lesion” There was also intense inflammatory infiltration and extensive areas of necrosis and hemorrhage, which allowed the morphological diagnosis of diffuse granulomatous rhinitis marked with intralesional yeasts compatible with Cryptococcus spp. Additionally, PCR was positive for C. gattii.Discussion: A case of nasal cryptococcosis caused by C. gattii in sheep in the state of Bahia is described for the first time, diagnosed using clinical-epidemiological, cytological, anatomopathological, and molecular data. Airborne infection occurs by inhaling encapsulated spores present in organic matter, such as decomposing vegetables and bird and bat excreta as observed on the farm where the outbreak occurred. It is important to highlight that sheep and goats affected by cryptococcosis can contaminate the environment, and it is prudent to consider animals with the disease as a risk to public health and eliminate them from the herd, given the costs of the few therapeutic options available on the market. It should be noted that in cases of tumor lesions, which are common in cryptococcosis, cytological examination should be recommended, mainly for screening and distinguishing neoplastic and inflammatory lesions. However, anatomopathological and molecular evaluations are essential for the differential diagnosis of other mycotic rhinitis, in particular, conidiobolomycosis and nasal pythiosis.

Diagnostic histological features of metastatic lymph nodes in adenosquamous carcinoma

Laryngeal adenosquamous carcinoma (ASC) is a rare malignancy, and the problems in true diagnosis of this entity are well known. In this paper, we describe a case of ASC and discuss its distinct histopathology in metastatic lymph nodes. A heavy smoker man presented with a progressive neck mass. Based on incisional biopsy, diagnosis of squamous cell carcinoma (SCC) was made and total laryngectomy with bilateral radical neck dissection was performed. Sections of primary tumor showed a well-differentiated SCC. However, lymph node metastases had a distinct feature of glandular structures. Further investigation with Mayer's mucicarmine stain confirmed the presence of rare scattered glands in primary tumor and the final diagnosis was changed to ASC. Adenosquamous carcinoma is a distinct entity and must be distinguished from the common SCC of larynx. As a result, when a lesion with diagnosis of squamous cell carcinoma shows evidence of glandular differentiation in a subsequent biopsy or in nodal metastases, more investigation is necessary. Moreover, the glandular structures in primary tumor, as in this case, may be rare and inconspicuous. Therefore, we suggest the precise evaluation of metastatic lymph nodes for any laryngeal tumor with diagnosis of SCC. This helps to make appropriate therapeutic decisions with respect to the aggressive behavior of ASC and its high propensity for lymph node metastasis.

Distribution of mucous cells on the body surface of Japanese flounder Paralichthys olivaceus

The distribution of mucous cells was examined in the skin on the ocular and blind sides of Japanese flounder Paralichthys olivaceus. Observations were performed on both body sides at the following regions: cheek, lower jaw (blind side), gill cover (ocular side), dorsal side, lateral line, belly and caudal peduncle. The mucous cells observed were elliptic and positively stained for periodic acid Schiff reaction and Mayer's mucicarmine and showed a higher density and larger size on the ocular side compared to the blind side. Low densities of mucous cells were observed on the lower jaw compared with other regions of the body. The depth of the crack located between scales was deeper on the ocular side than the blind side, which might reflect total epidermis area and total number of mucous cells. Bacterial infection elucidated some information on the effect on the density and size of mucous cells, where the density and size decreased slightly after infection. Only the lower jaw, however, showed an increased number of mucous cells. The results show that the potential of skin to secrete mucus is higher on the ocular than on the blind side and bacterial infection decreases mucous secretion.

Unusual morphologies of Cryptococcus spp. in tissue specimens: report of 10 cases

Ten cases of cryptococcosis due to unusual microscopic forms of Cryptococcus sp. observed over a twenty-eight year period (1981-2009) are presented. The most important clinicopathological and laboratory data are tabulated. The uncommon forms of cryptococcal cells given are: structures resembling germ tube (one case), chains of budding yeasts (one case), pseudohyphae (two cases) and nonencapsulated yeast-like organisms (eight cases). The diagnosis was based on the histopathological findings. The causative organism was isolated and identified in seven cases; five were due to C. neoformans, and two to C. gattii. In addition, the importance of using staining histochemical techniques - Grocott's silver stain (GMS), Mayer's mucicarmine stain (MM) and Fontana-Masson stain (FM) - in the diagnosis of cryptococcosis is argued.

Histopathology, serology and cultures in the diagnosis of cryptococcosis

Cryptococcosis is one of the most common opportunistic fungal infections in patients with acquired immunodeficiency syndrome (AIDS). We report 13 cases of cryptococcal infection based on histopathology, serology and cultures. Epidemiological analysis, histochemical techniques of hematoxilin and eosin (HE) and Grocot's silver (GMS), as well special histochemical techniques such as Mayer's mucicarmine (MM) and Fontana-Masson (FM), cryptococcal antigen test (CrAg) and isolation on fungal media: Sabouraud's (SAB), brain-heart infusion agar (BHI) and canavanine-glycine-bromothymol blue (CGB) agar were analyzed. Unsatisfactory staining results by MM stain associated to negative titers by CrAg test, which FM stain confirmed that capsule-deficient Cryptococcus infections were observed in four cases. Eight isolated cases were identified as follows: six cases were infection with Cryptococcus neoformans and two cases were Cryptococcus gattii.

Nonneoplastic signet-ring cells in the gallbladder and uterine cervix. A potential source of overdiagnosis

We describe 3 cases of nonneoplastic signet-ring cell change in ulcerated mucosa, 2 of them in the gallbladder and 1 in an endocervical polyp. In the gallbladder cases, there were focal collections of signet-ring cells both on the mucosal surface and within the lumen of tubules, whereas in the endocervical polyp, the signet-ring cell aggregates were entirely confined to the mucosal surface. In all 3 cases, the signet-ring cells were positive for Mayer's mucicarmine and immunoreactive for keratin AE1/AE3. The lack of nuclear atypicality, the arrangement in superficial and intraluminal nests, and the admixture with histiocytes and other inflammatory cells are in keeping with the interpretation that the signet-ring cells are disrupted mucosal goblet cells exhibiting hyperplastic and degenerative changes. A review of the literature disclosed only other 2 previously reported cases of benign signet-ring cell changes in the gallbladder and none--to the best of our knowledge--in an endocervical polyp. Awareness of this phenomenon is of importance to avoid a potential overdiagnosis of signet-ring cell adenocarcinoma.

The foamy variant of pancreatic intraepithelial neoplasia

Foamy gland adenocarcinoma is a variant of pancreatic ductal carcinoma, whose precursor has not been described. We describe here the morphologic and immunohistochemical features of the pancreatic intraepithelial neoplasia (PanIN) lesions associated with invasive foamy pancreatic adenocarcinoma. The staining properties and morphologic and immunohistochemical features of 3 foamy PanIN lesions were compared with those of 7 pancreatic foamy gland adenocarcinomas. Hematoxylin and eosin, Mayer mucicarmine, periodic acid-Schiff, and Alcian blue stains were available for review in all cases. Immunohistochemical labeling for cytokeratin (CK)7, CK20, carcinoembryonic antigen polyclonal, MUC1, MUC2, CDX2, p53, and cyclin D1 was performed. The PanIN-1 lesions were found in the nonneoplastic pancreas and were similar to the PanIN-1 lesions of ordinary pancreatic ductal carcinoma. The PanIN-2 and -3 lesions were recognized immediately adjacent to or within the invasive foamy gland carcinoma. In these lesions, small or markedly dilated ducts were lined by cuboidal and columnar dysplastic nonfoamy cells and foamy cells. Hobnail cells were present in 2 cases. The PanIN-1, 2, and 3 lesions and the invasive foamy gland adenocarcinomas stained with mucicarmine, periodic acid-Schiff, and Alcian blue. The 3 PanIN-2 and -3 lesions and all 7 invasive foamy adenocarcinomas labeled with CK7, carcinoembryonic antigen polyclonal, and MUC1, whereas only 2 PanIN-2 and -3 lesions and 5 invasive adenocarcinomas showed immunoreactivity for cyclin D1 and p53. Three distinctive foamy PanIN lesions were identified within 7 invasive foamy gland pancreatic adenocarcinomas. The gradual progression of cytological and architectural abnormalities of the PanIN lesions from PanIN-1 to PanIN-3 excludes neoplastic ductal spread. These foamy PanIN lesions probably represent cancer precursors.

Sclerosing mucoepidermoid carcinoma of the salivary glands

Mucoepidermoid carcinoma (MEC) comprises approximately 30% of all salivary gland malignancies, making it the most common malignant tumor of the salivary glands. Multiple histologic variants with a wide range of differentiation have been described. Sclerosing MEC (SMEC) is a rare subtype that may be misdiagnosed as a benign reactive condition or low-grade non-SMEC malignancy. We report 4 cases of SMEC and evaluated them with Her-2/neu and MIB-1 to determine whether an association exists between the histologic grade and immunohistochemical findings. In 3 cases, histologic examination demonstrated relatively well-circumscribed, nonencapsulated tumors composed of extensive central sclerosis with keloid-like stroma and scattered epithelial islands of low-grade MEC. In the fourth case, the tumor showed similar sclerotic stroma; but the epithelial component was of intermediate grade. In all 4 cases, eosinophils and neutrophils were part of the inflammatory infiltrate; and the edges were surrounded by lymphoid tissue, with germinal center formation and residual epithelial islands. A Mayer mucicarmine stain revealed abundant intracytoplasmic mucin. We found MIB-1 labeling indices of 5% or less in cases 1, 2, and 3 and 12% in case 4, suggesting an association between MIB-1 index and tumor grade. The tumors were negative for Her-2/neu in all 4 cases. The latter seems to bear no relationship to tumor grade.

Invasive Cryptococcosis and Adalimumab Treatment

Invasive Cryptococcosis and Adalimumab Treatment

Nodular hyperplasia of Bartholin’s gland

There has been very little mention of benign solid lesions of the Bartholin's gland (BG) in pathology and gynaecology textbooks, and very few cases have been reported in the literature. Among these lesions, the distinction between nodular hyperplasia (NH) and adenoma has not been well defined. We report ten cases of NH of the BG, describe their clinicopathological, immunohistochemical and ultrastructural findings, and review the literature. We examined retrospectively all lesions involving BGs from our surgical pathology records from 1990 to 2004 with emphasis on NH. To separate NH from adenoma, we applied the criteria proposed by Koenig and Tavassoli. Special stains (PAS with and without prior digestion, Mayer's mucicarmine and Alcian blue with and without hyaluronidase) and immunohistochemistry (CAM5.2, AE1/AE3, HMWK, monoclonal CEA, EMA, ER, PR, ALA, SMA, Ki-67, p53 and polyclonal CEA) were performed on NHs. Two cases were examined ultrastructurally. Using specific criteria, ten cases (age range 23-45 years; mean 36.1) of NH were identified, two of which were diagnosed previously as adenoma, but re-classified as NH. Clinically, these lesions were described either as Bartholin's duct cysts (BDCs) or vulvar lumps. Grossly, NHs were solid, tan and unencapsulated, measuring 12.5-45.0 mm in maximum dimension (mean 23.8). Histologically, the NHs were composed of a proliferation of mucus-secreting acini with preservation of the normal duct-to-acinar relationship. Chronic inflammation and squamous metaplasia were present. Eight lesions focally involved the surgical margins. Intracytoplasmic and intra-luminal secretions were positive for PAS with and without digestion, Alcian blue with and without hyaluronidase and mucicarmine. All lesions showed positive staining for CAM5.2, AE1/AE3, HMWK, EMA, and polyclonal CEA. There was negative staining for Ki-67, ER, PR, ALA, p53 and monoclonal CEA. Periacinar myoepithelial cells stained for SMA. Ultrastructurally, the findings included abundant intracytoplasmic secretory granules, granulofibrillar bodies, prominent Golgi and ribosomes. Myoepithelial cells were identified. There was no tumour recurrence or malignant transformation in eight patients with clinical follow-up. NH of the BG is a rare lesion with benign behaviour. It is a distinct entity and can be separated histologically from an adenoma.

Microscopical examination of the localisation patterns of two novel rhodamine derivatives in normal and neoplastic colonic mucosa.

Tissue characterisation by fluorescence imaging, using exogenous fluorophores, is a promising method for cancer detection. Histochemical alterations in the composition of mucins, when neoplastic transformations occur, could be exploited to derive more selective fluoroprobes indicative of early malignant transformation. The aim of this work was to develop and examine tumour selective fluoroprobes for colon cancer diagnosis, as well as to determine the morphological components where selective dye accumulation has occurred. Two novel fluoroprobes: rhodamine B-L-leucine amide and rhodamine B-phenylboronic acid were synthesised and examined together with Mayer's mucicarmine, alexa 350-wheat germ agglutinin (WGA) and tetramethyl rhodamine-concanavalin A (ConA). Fluorescence microscopy studies were performed with deparaffinised human colon sections, using an epifluorescence microscope equipped with a colour CCD camera. The intense accumulation of the novel fluoroprobes was localised in the amorphous material in the lumen of neoplastic crypts. To gain insight into the localisation patterns, mucicarmine, alexa 350-WGA and tetramethyl rhodamine-ConA were used. Alexa 350-WGA reacted primarily with mucin secreted in the malignant crypt lumen suggesting that this material is rich in sialic acid and N-acetylglucosaminyl residues. These derivatives clearly and consistently distinguished non-neoplastic from neoplastic human colon tissue sections. The intense accumulation at the altered mucins indicates that they could be used as fluoroprobes of biochemical alterations for carcinoma detection.

Glucose Transporter Glut-1 Is of Limited Value for Detecting Breast Carcinoma in Serous Effusions

Diagnosing breast carcinoma that has metastasized to body cavity fluids can be difficult. Recently, immunostaining for the facultative glucose transporter Glut-1 has been described as a sensitive and specific means of detecting carcinomas in effusions. However, only five cases of breast carcinoma were studied. We examined Glut-1 specifically as a means of detecting breast carcinoma in effusion cytology. Using avidin-biotin immunocytochemistry, cell block material from 31 cases of breast carcinoma metastatic to body cavity effusions and 33 cases of benign effusions were studied. All cases were immunostained with the Glut-1 antibody. An additional set of slides from these same cases was stained for mucin using the Mayer's mucicarmine technique. Slides were graded for percentage of cells exhibiting immunoreactivity for Glut-1 or for the presence of mucin. Results of staining for both Glut-1 alone and in combination with mucicarmine were compared between the benign and malignant groups. Of the breast cancer cases, 19 of 31 (61%) were immunoreactive for Glut-1, and 25 of 31 (81%) were positive for either Glut-1 or mucicarmine. One of the 33 (3%) benign cases was immunoreactive for Glut-1, and none were positive for mucin. These data suggest that using Glut-1 as a single immunostain or in conjunction with mucicarmine is a specific but modestly sensitive means of detecting breast carcinoma in this cytologic setting.

Evaluation of histochemical methods for the detection of intracytoplasmic mucin in serous effusions.

We have studied 25 serous effusions containing definitive morphologic evidence of adenocarcinoma to evaluate the ability of two mucin stains (Mayer's mucicarmine, D-PAS) to detect intracytoplasmic mucin in both cytologic (cytospin) and corresponding histological (cell block) preparations. Mucicarmine stain was positive in six of 25 (24%) cytospins and 13 of 25 (52%) cell blocks. D-PAS was positive in 19 of 25 (76%) cytospins and 20 of 25 (80%) cell blocks. Eight cases were identified which showed mucicarmine positivity in the cell block but not the corresponding cytospin; prolonging incubation time resulted in a positive mucicarmine in cytospin preparations for seven of these cases. We conclude that: (i) D-PAS is a more sensitive stain for the detection of intracytoplasmic mucin in all preparations; (ii) mucicarmine shows preferential staining for cell blocks; (iii) alterations in the staining protocol may permit mucin detection by mucicarmine staining in cytologic preparations in a significant number of cases.

Papillary (chromophil) renal cell carcinoma with mutinous secretion

We investigated the presence of mucin in a series of 20 papillary renal cell carcinomas. Acid mucin was present in three cases (15%), in which basophilic mucin secretion was evident with hematoxylin-eosin. This secretion reacted positively with Mayer's mucicarmine, Alcian blue and high-iron diamine, but was negative for PAS in all the cases, indicating the presence of sulphomucins. We describe two different types of mucin secretion: luminal and intracytoplasmic. The secretion was abundant, diffuse or extensive, luminal in two cases and intracellular in numerous scattered tumor cells in one case. All tumors were < 3 cm in diameter (low grade, stage I). In the three mucin-secreting papillary tumors mentioned above, the immunohistochemical and lectin studies indicate both a proximal and a distal tubular staining pattern. Mucinous secretion in these tumors can be ascribed either to modulation or direct metaplasia of the tumor epithelium. Mucin production, despite its low frequency, can be considered an additional feature of papillary renal cell carcinoma. Thus, the presence of luminal or intracytoplasmic mucin deposition does not exclude papillary renal cell carcinoma from the differential diagnosis in cases of intra- or extrarenal carcinomas.

Cytologic diagnosis of Cryptococcus neoformans in HIV-positive patients.

To present experience with the cytologic diagnosis of pulmonary cryptococcosis in bronchial washings and bronchoalveolar lavage in human immunodeficiency virus (HIV)-positive patients in Thailand. The study group consisted of 54 HIV-positive patients admitted to the Faculty of Medicine, Srinagarind Hospital, during the period January 1992-June 1994. Bronchial washing and bronchoalveolar lavage specimens were studied for Cryptococcus neoformans by routine Papanicolaou and special staining methods. The Cryptococcus organism, an encapsulated yeast with a clear halo on Papanicolaou-stained smears, was found both extracellularly and in histiocytes in 4 of 54 (7.4%) cases. A definitive diagnosis was confirmed by positive staining of the capsular mucopolysaccharides with periodic acid-Schiff and Mayer's mucicarmine stain and of the cell wall with methenamine silver. Lung biopsy was performed in one case and revealed cryptococcosis. This study underscores the usefulness of cytologic screening in the detection of this opportunistic pulmonary infection. The cytopathologist and cytotechnologist should be alert for the presence of Cryptococcus in cytologic specimens from HIV-positive patients.

EOSINOPHILIC GLOBULES IN KAPOSI'S SARCOMA

Of the 12 cases, 2 (17%) showed eosinophilic globules in the typical cutaneous type of Kaposi's sarcoma. The globules were stained with periodic acid-Schiff (PAS), periodic acid-Schiff reagent after diastase digestion, and phosphotungstic acid hematoxylin (PTAH), but were not stained with Mayer's mucicarmine, and alcian blue. As the results, these globules might be glycoprotein. The shape of the globules was very similar to glycoprotein globules of yolk sac tumor (endodermal sinus tumor) in the tissue of the ovary and testis. In the yolk sac tumor, similar globules are stained with alpha-fetoprotein, beta-subunit of human chorionic gonadotropin, and alpha-1-antitrypsin using immunohistochemical techniques. Immunoperoxidase investigations were done with antibodies to alpha-fetoprotein, beta-subunit of human chorionic gonadotropin, alpha-1-antitrypsin, and carcinoembryonic antigen in the eosinophilic globules of Kaposi's sarcoma, but these antigens were detected in the globules. The morphogenesis of the glycoprotein globules is not clear yet. A better understanding of the source of globules in Kaposi's sarcoma awaits further research.

Endocervical and endometrial adenocarcinoma: an immunoperoxidase and histochemical study.

Carcinoembryonic antigen (CEA) has been demonstrated in endocervical but not in endometrial adenocarcinomas, suggesting a valuable differentiating feature. Using an immunoperoxidase technique for CEA, alcian blue (AB)-periodic acid-Schiff stain for acidic and neutral mucosubstances, respectively, and Mayer's mucicarmine for epithelial mucosubstances, 13 endocervical and 21 endometrial adenocarcinomas were studied to delineate differentiating criteria. CEA was present in all endocervical adenocarcinomas with strongly positive immunostaining in 69%, diffusely throughout the tumor in 77%, intracellularly in 77%, in luminal secretions in 39%, and at the cell surface in 31%. Only 11 (52%) endometrial adenocarcinomas contained CEA, 82% with weak immunostaining, 64% focally, 82% at the cell surface and 36% intracellularly. Mucin was present in 12 (92%) endocervical lesions with AB-positive intracellular and luminal secretion in 83%, strong positivity in 42%, and a diffuse distribution in 60% with all three stains. Moderate or small amounts of mucin were present in 19 (91%) endometrial lesions, focally in 80%, in luminal secretions in 94%, and intracellularly in 42%. Thus, the majority of endocervical adenocarcinomas showed abundant diffusely distributed intracellular CEA and mucin, and mucinous luminal secretions. Half of the endometrial adenocarcinomas contained focal small amounts of apical surface CEA. Mucin was present focally particularly in luminal secretions.

Fine Structure of the Outer Epidermis of the Viviparous Monogenean Gyrodactylus sp. from the Skin of Gasterosteus aculeatus

The fine structure of the covering layer of Gyrodactylus sp., an unusual monogenean with a peculiar kind of viviparous development, was studied for comparison with the outer layer of more conventional monogeneans which have a ciliated oncomiracidium larva. The outer layer of Gyrodactylus was found to be a syncytial epidermis like that of other monogeneans, digeneans, and cestodes, but unlike these other parasitic platyhelminths the superficial epidermis of Gyrodactylus does not appear to connect with nucleated regions (inner tegument) situated in the parenchyma. As full-term but unborn embryo worms have a nucleated superficial epidermis it seems likely that the adult epidermis either contains very few nuclei which would be difficult to observe, or that nuclei once present may have degenerated. The adult epidermis contains few ribosomes but Golgi may be present. The nucleated epidermis of embryo Gyrodactylus is packed with ribosomes; and vesicles which may contain mucoor glycoproteins are already present in the epidermis of late embryos. Lamellate inclusions consisting of membrane stacks or whorls have been found in the epidermis of both embryos and adults. These could be membrane storage depots although the possibility of their being fixation artifacts cannot be completely eliminated. Work with ferritin suggests that the outer layer is extremely impermeable to this colloid although in the gut ferritin is actively pinocytosed. The covering layer of the marine ectoparasitic monogeneans, Entobdella soleae and Acanthocotyle elegans, has recently been shown to be a syncytial cytoplasmic layer which connects with parenchymally situated epidermal cell bodies (Lyons, 1970) and is very like the outer layer of the endoparasitic digeneans and cestodes. A survey of the representatives of various monogenean groups (Lyons, 1968) suggests that this kind of outer layer occurs throughout monogeneans. Gyrodactylus is an unusual monogenean because it reproduces in a specialized way. The free-swimming larval stage found in all other groups of monogeneans is absent. The worms are viviparous and reproduce by a peculiar form of polyembryony such that one fertilized ovum eventually gives rise to four individuals which develop within one another and within the adult worm at different rates (Bychowsky, 1957; Dogiel, 1964; Kathariner, 1904). Work on the fine structure of the outer layer of Gyrodactylus suggested that this was unlike that found in other groups of monogeneans and that the differences could be related to the specialized kind of reproduction found in this group. MATERIALS AND METHODS Specimens of Gyrodactylus sp. (as yet unnamed and undescribed) were obtained from the skin and Received for publication 3 March 1970. fins of three-spined sticklebacks from Emmanuel College pond, Cambridge. Worms fixed and flattened in 10% formalin for 2 days were stained with alcian blue for acid mucopolysaccharides, the periodic acid-Schiff method for mucopolysaccharides and carbohydrates (Pearse, 1961), and Mayer's mucicarmine for mucus (Gomori, 1952). Whole worms were fixed for 1 to 1.5 hr in 2.5% glutaraldehyde in cacodylate buffer at pH 7.2 and a total molarity of 0.1 M. Material was postfixed at 4 C for 1 hr in 1% osmium tetroxide (Rosenbluth, 1965). Following rapid dehydration in cold ethanol, material was embedded in Araldite (Richardson, Jarrett, and Finke, 1960). Sections cut with glass knives on a Cambridge Instrument Co. ultramicrotome were mounted on uncoated copper grids and stained with a saturated solution of uranyl acetate in 50% ethanol, or for low-power work, with 30 to 50% uranyl acetate in absolute methanol for 3 min followed by Reynolds' lead citrate. The sections were viewed on AEI EM 6B and Philips EM 200 electron microscopes. In order to compare the possible ability of the epidermis and gut respectively to transport ferritin, specimens of Gyrodactylus were incubated in a 2% ferritin solution in tap water for 0.5 hr before being prepared for electron microscopy.

Histochemical observations on the cercariae of Posthodiplostomum minimum

The cercariae of Posthodiplostomum minimum, collected from freshwater snails in Georgia, were subjected to various histochemical tests to determine the nature of the contents of the gland cells. Three pairs of gland cells were obvious within the cercariae. One pair was preacetabular in position, and two pairs were postacetabular. The ducts from all the glands opened at the extreme anterior portion of the animal, a member of each pair opening on either side of the mouth. The granular contents of the gland cells stained positively with the Schiff reaction. The tests for tyrosine, arginine, and basic proteins were likewise positive. In addition, the results of other histochemical tests, such as the Alcian blue test and Mayer's mucicarmine, indicated the presence of acid mucopolysaccharides. It was apparent, therefore, that two types of secretions were in the gland cells: a carbohydrate-protein complex, probably a glycoprotein, as well as an acid mucopolysaccharide. The major carbohydrate found within the parenchyma was glycogen. Alkaline glycerophosphatase was limited to the bipartite excretory bladder. The presence of deoxyribonucleic acid was limited to the nuclei of the parenchymal cells and to the nuclei of the gland cells. Ribonucleic acid was present in small amounts in the contents of the gland cells and was the major nucleic acid in the cytoplasm of the parenchymal tissue. A brief description of the morphology of the gland cells was given.