Ziehl-Neelsen Method Research Articles

Evaluation of a safe sputum processing method for detecting tuberculosis.

To evaluate a safe sputum processing method for detection of tuberculosis in developing countries. A sample processing method was developed in which acid fast bacilli were killed with 1% sodium hypochlorite and concentrated by flotation on a layer of xylene before staining by the Ziehl Neelsen or auramine O methods. Best results were obtained by auramine O staining after flotation. Staining by the Ziehl Neelsen method after flotation gave better results than direct Ziehl Neelsen staining without flotation. The flotation method with Ziehl Neelsen staining offers advantages for smear preparation in the tuberculosis control programmes of developing countries.

Immunohistochemical studies on mycobacteria in tuberculous necrotizing granulomas

For the purpose to know the pathogenesis of caseous necrosis in tuberculous lesion, authors compared the presence and the number of mycobacteria in the pulmonary tubercles of various histological types. Forty pulmonary tubercles obtained from 16 patients (9 males, 7 females; mean age 51) were examined. By Ziehl-Neelsen (ZN) method, acid-fast bacilli were detected in 40% of 5 proliferative, in 12.5% of 24 productive and none of 11 sclerotic granulomas. By immunohistological staining with anti-BCG antiserum, positive staining was observed in 60%, 87.5% and 91% of each type of granuloma, respectively. Types of necrosis were classified by silver-staining as necrosis after exudative reactions (EN), necrosis after productive reactions (PN) and filled materials after softening and liquification of necrotic lesions (LN). EN was observed in 24 tubercles, and mycobacteria were stained in 8% of EN by ZN method and in 75% by immunohistological method. PN was observed in 21 tubercles, and mycobacteria were detected in none and 5% of such type of necrosis by respective method. LN was observed in 28 tubercles, and mycobacteria were detected in 11% and 71% of these lesions by respective method. So, positive ratio by immunochemical method was significantly higher in EN and LN than in PN. Further, mycobacteria were stained with anti-BCG antiserum as granules and the number of such granules were much more numerous in EN and LN than in PN (p < 0.01). From these results, it was suggested that necrosis might occur by different mechanisms in exudative reactions and in productive reactions.

Prevalence of cryptosporidiosis in HIV-infected patients with diarrhoeal illness.

Protozoans of the genus Cryptosporidium may cause serious diarrhoeal illness in immunocompromised hosts and especially in HIV-infected patients. In this study we have evaluated the frequency of Cryptosporidium in stools of 51 HIV-infected patients with diarrhoea. Laboratory diagnosis of cryptosporidiosis was performed of faecal samples concentrated by a formalin-ether sedimentation technique and stained by a modified cold Ziehl-Neelsen method. Results demonstrated that 17 (33.3%) of these patients were infected with Cryptosporidium. Moreover, Cryptosporidium infection was the first clinical marker of AIDS in 7 cases. Our data show that the prevalence of this parasitosis in HIV-infected people seems to be higher in our region (Apulia, South Italy), compared to other areas of the world.

原発性肝放線菌症の1例

We present a case of a 71-year-old female with actinomycosis in the liver, which is a rare region to be primarily affected with actinomycosis. The diagnosis was done histopathologically with a partially resected liver specimen taken during surgery for choledocholithiasis. There were no clinical signs of Actinomyces infection before surgery. The hepatic lesion was a 2 cm sized nodule with histological appearances of abscess-forming suppurative inflammation with fibrosis, in which eosinophilic radiate granules with peripheral clubs were found. The Brown-Brenn stain showed Gram-positive branched filamentous bacilli, which were revealed as acid-fastness by the Ziehl-Neelsen method of Putt's modification. These findings were considered to be consistent with actinomycosis of the liver.

A simple staining method for the identification of chlamydial elementary bodies in the fetal membranes of sheep affected by ovine enzootic abortion

A dark-ground methylene blue (DGMB) staining method was used to demonstrate chlamydial elementary bodies in fetal membranes of sheep affected by Chlamydia psittaci. Before evaluation on material from clinically affected animals, the DGMB method was compared with modified Ziehl-Neelsen (MZN) and dark-ground Giemsa (DGG) staining methods for its ability to demonstrate chlamydial elementary bodies in hen's eggs which had been experimentally infected with C. psittaci. DGMB was more specific in its staining of chlamydial elementary bodies than DGG or MZN. The DGMB method was found to be a more reliable technique for the examination of fetal membranes from sheep affected with C. psittaci than DGG or MZN. Those samples diagnosed as positive using the DGMB showed a good correlation with those diagnosed as positive on macroscopic examination.

Differential Staining of Schistosome Ova in Biopsies

The significance of identifying the infecting schistosome species has been highlighted recently by the availability of specific chemotherapeutic agents and an awareness that the tissue response – and therefore the prognosis – may be species-dependent. Moreover, human interference with the ecological systems in parts of the developing world has modified the epidemiological map of schistosomiasis resulting in a rising incidence of mixed infections. Differentiation between the ova of Schistosoma mansoni and S. haematobium, the two species commonly seen in immigrants to Kuwait and to other non-endemic areas of the world, usually poses no problems in urinary and faecal specimens. However, in histopathological samples, their distinctive morphological features cannot often be assessed reliably. We obtained satisfactory differentiation by using a modified Ziehl-Neelsen method on 81 routinely fixed and processed biopsy samples. Moreover, on destained sections previously stained with haematoxylin and eosin, the differences were equally evident. Details of the method and its applications both in routine histopathological practice and for research are discussed.

Disseminated Mycobacterium Avium Serotype 1 Infection in a Seven-Month-Old Cat

A 7-month-old, neutered, male, domestic shorthair cat was presented for examination to a practicing veterinarian after 2 weeks of anorexia and unspecified treatment for “chronic respiratory disease.” Physical and hematologic examinations revealed generalized lymphadenopathy, fever (39.7 C), and a corrected total blood leukocyte count of 17,329/μ1 with 27 nucleated red blood cells/100 leukocytes. Blood packed cell volume was 17%, and plasma contained 6.5 g/d1 total protein. After the animal had shown nonspecific signs for an additional week, smears made from aspirated bone marrow and a popliteal lymph node collected by biopsy and fixed in formalin were referred to the College of Veterinary Medicine at Iowa State University (ISU) for microscopic evaluation, and the cat was returned to its owner. Following a histologic diagnosis of disseminated mycobacterial disease, the cat was rehospitalized at the referring clinic for further observation and treatment. No cutaneous lesions were found. After consultation with the owner, the cat was euthanatized, necropsy was performed by the referring veterinarian, and tissue specimens were collected and submitted to ISU for further laboratory examination. Samples of lungs, liver, spleen, intestine, and tracheobronchial and mesenteric lymph nodes were received both frozen and in formalin. The remaining popliteal lymph node was received frozen, and bone marrow from a femur was received fixed in formalin. Blood had been collected in serum and anticoagulant (ethylenediaminetetraacetic acid) tubes prior to euthanasia. This report describes disseminated Mycobacterium avium serotype 1 infection of a young cat in the absence of cutaneous lesions or evidence of antecedent immunosuppressive disease or treatment. At necropsy, mandibular, ileocecal, and tracheobronchial lymph nodes were reportedly “very enlarged,” and the lungs contained nodular densities throughout. The fixed ileocecal and tracheobronchial lymph nodes and lung nodules measured 1.0, 1.5, and 0.5 cm in diameter, respectively. Bone marrow smears revealed adequate megakaryocytes, myeloid hyperplasia, and erythroid hypoplasia. No atypical cells were noted, but fine, elongate, refractile forms, unstained by Wright’s stain and frequently aggregated in clusters, were seen in the cytoplasm of mononuclear cells and large multinucleate cells (Fig. 1). Acid-fast staining by the Ziehl-Neelsen method (ZN) showed abundant, 4-6 μm, slender, acidfast (beaded) organisms in the same cells. Peripheral blood smears and whole blood revealed anemia (hemoglobin 5.6 g/dl packed cell volume 18%, red blood cell count 2,850,000/ μl), leukopenia (total corrected leukocyte count 4,700/μ1), and lymphopenia (1,175/μ1). Serum contained 6.6 g/d1 of total

Accumulation of pigment granules in lacrymal gland epithelium in practolol-treated beagle dogs

A 6-month oral toxicity test of practolol was carried out in beagle dogs as a reference control for a newly developed beta blocker. No significant drug-induced changes were detected in any animals by various ophthalmological examinations such as ERG, tear flow, lysozymal activity in tears, etc. However, an unusual pathological change was detected in the lacrymal gland of all five dogs treated with practolol and not in control animals. Macroscopically, the lacrymal glands assumed a blackish brown to deep black colour on both the outside and the cut surface. Microscopically, fine, dark-brown pigment granules were present in the apical and supra-nuclear portion of the cytoplasm of predominantly serosal type epithelial cells. These pigments reacted positively to Schmorl's method for lipofuscin, but gave a negative PAS reaction for polysaccharide, Prussian blue for iron and Ziehl-Neelsen method for ceroid pigment. They were detected as membrane-bound electron-dense bodies by electron microscopy. Similar pigments were also deposited in the cytoplasm of the apocrine sweat gland. Although the mechanism of the accumulation of these granules is far from clear, concentration of practolol in the lacrymal gland is considered to be very closely related to the presence of these granules. A possible mechanism for ocular toxicity by practolol, involving this change, is discussed.

A cold staining method for acid-fast bacilli

The Ziehl-Neelsen method is probably the best known and most frequently used procedure for staining tubercle bacilli. The method requires controlled heating for its success. However, in developing countries, such as India, where most laboratories rely mainly on spirit lamps as a source of heat, the Ziehl-Neelsen method often cannot be carried out because rectified spirit is difficult to obtain. The study describes a cold staining technique that uses the same staining solutions as the conventional Ziehl-Neelsen method. For direct smears, the correlation of results of the cold staining procedure with those of the Ziehl-Neelsen method was 97% and for concentrated smears was 99%. The method described is suitable for use in basically equipped laboratories.

Cryptosporidiosis in southern Sweden.

Two different groups of patients with diarrhoea, altogether 1,478 individuals, were examined for cryptosporidium oocysts. The technique used was feces concentration and staining according to a modified Ziehl-Neelsen method. 20 cases of cryptosporidiosis (3%) were found among 698 consecutive patients with acute gastroenteritis. In feces samples sent for parasitological examination from 780 patients, cryptosporidia were found on 9 occasions (1%). None of 519 healthy persons excreted cryptosporidia. 19/29 patients with cryptosporidiosis had recently been abroad and in 8 of these additional enteric pathogens were found. The median duration of diarrhoea was 14 days. All patients except 1 were cryptosporidia-negative in faeces within 2 months. An immunosuppressed patient excreted cryptosporidia for 14 months.

Bulk staining of sputum by the Ziehl-Neelsen method

A trial of the Ziehl-Neelsen staining method employing bulk decolorization, rinsing and counter staining yielded reliable results. Of 110 patients who proved to be positive by direct microscopy, cross-contamination could not be proved in any. The advantage of the mass method is its economy.

Negative staining method with nigrosin for the detection of cryptosporidial oocysts: a comparative study

A comparison was made between two methods for the detection of cryptosporidial oocysts in human faecal sediments of formalin-ether concentrates: a cover-slipped, wet method (containing Gram's iodine) and an air-dried, negative staining method using nigrosin solution. A modified Ziehl-Neelsen technique was used as a reference method. The negative staining method with nigrosin gave a positive diagnosis more often than the cover-slipped wet method. A comparison of the nigrosin method with the modified Ziehl-Neelsen method gave almost identical results. Restaining the nigrosin slides with the modified Ziehl-Neelsen technique showed that the round, refractile bodies were cryptosporidial occysts.

Identification of mycobacteria by smear examination of the culture

Mycobacteria can be tentatively identified by the arrangement of the bacilli on smears made from the growth and stained by the Ziehl-Neelsen method. The basis of this method is the presence or absence of cords with or without loose bacilli around. Agreement of a high degree (92.7 % to 97.0 %) was obtained between the identification of mycobacteria by this smear technique and by the niacin test. It is recommended that this simple procedure may be used for preliminary identification, especially in developing countries.

Diatoms in the Gills of the Commercial White Shrimp

A white shrimp from Galveston, Texas, is the first reported case of a crustacean internally infected by a diatom. Even though more than one species occurred in debris on and between gill filaments, only individuals of Amphora sp. occurred within gills. To determine if a related diatom would easily reproduce within the shrimp and cause. a hostresponse similar to that observed, we injected cultured specimens of A. coffaeformis into white shrimp. Under the experimental conditions, individuals of that species did not divide, but they elicited an extensive melanistic host-response. Gills of a single specimen of the white shrimp Penaeus setiferus from Galveston, Texas, harbored numerous melanistic regions. This particular specimen, the first crustacean reported to be infected in the hemocoel with algae, had been collected from the natural environment and maintained along with others during a project dealing with shrimp maturation. Soon after death, it was fixed in Carnoy’s I1 solution and sent to us for identification of the agent or agents eliciting the melanistic response. Jorge Leong, who forwarded the specimen, noted no similarly affected shrimp at his laboratory at that time. Gross examination of a stained preparation (Van Cleave’s hematoxylin) of a few gills revealed clusters of diatoms associated with deposits of presumed melanin. These clusters consisted of Amphora sp. On the other hand, individuals of Amphora sp., Nitzschia spp., and Achanthes spp. (including A. exigua) occurred in debris on and between filaments. Additionally, an apostomatid ciliate occurred within a few filaments. Sectioned material showed several sites, primarily in the tips of filaments, where hemocytes had apparently encircled diatoms. In the voluminous hemolymph-filled space of the afferent channel of one gill adjacent to its lamellar junctions, Amphora sp. occurred in large clusters. Stained internal structures within the diatoms suggest that most were alive at the time of fixation. Apparently Amphora sp. grew and reproduced within the shrimp, since individual organisms in the afferent channel ranged from small to large and exhibited a variety of shapes. A heterogeneous debris-like substance surrounded most of the internal clusters of diatoms, possibly resulting from their metabolic wastes. The substance contrasted with the lightly stained hemolymph by staining red and violet with Taylor’s technique for bacteria and blue and violet with the Ziehl-Neelsen method for bacteria (Luna 1968). In contrast to that in most shrimp, the hemolymph in this particular shrimp had a fibrinous consistency. Manuscript received May 12, 1980; accepted July 2, 1980. In an effort to determine if diatoms easily reproduce in the white shrimp, we injected saline with cultured Amphora coffaeformis into either the hemocoel or abdominal musculature of 50 shrimp. We used A. coffaeformis since it closely resembled the unidentified species present in the shrimp from Texas. Within an hour, most injected diatoms concentrated in the gill region. By 24-hour postinoculum (PI), we detected melanization as yellowish-brown foci. These regions acquired a dark color by day 2. By day 4 they became more numerous, larger, and darker. Seldom did more than a few individual diatoms occur together, suggesting their failure to reproduce. Nevertheless, the inoculated shrimp died sooner than saline-injected counterparts used as controls. When only a few individual diatoms were introduced into shrimp in a follow-up experiment, no aggregaiions became apparent after 7, 14, or 21 days PI. We conclude that the single infected shrimp from Texas probably represents an abnormal or accidental case. Possibly, recently eaten diatoms passed or were forced through a damaged or filled alimentary tract. In any event, the host apparently encapsulated diatoms at a slower rate than some individuals could multiply. In the experimental shrimp a factor such as the strain of diatom, health or resistance of shrimp, or other influence may have inhibited reproduction of the diatom. Once certain diatom species gain entrance into a crustacean’s hemocoel, hemolymph might provide a good culture medium. Amphora spp. are known to be remarkably versatile in their requirements for growth. Cooksey and Chansang (1 976) reported different requirements for three different heterotrophic cultures of Amphora spp. Moreover, some species withstand harsh environments. Amphora coffaeformis adjusts by establishing resting cells which can rapidly reestablish to the logarithmic stage given favorable conditions (Anderson 1975). Melanization and the cellular inflammatory response in penaeids and other arthropods have already been documented. Lightner and Redman (1977) reported that a variety of pathological conditions, organisms, heavy metals, and

Staining of mast cell granules by the Ziehl-Neelsen method and differential diagnosis of malignant dermal tumours in the dog.

Staining of mast cell granules by the Ziehl-Neelsen method and differential diagnosis of malignant dermal tumours in the dog.

マウスによる人らい菌増殖の試み

The technical procedures of experiment were explained briefly in Table 1. Bacterial suspension prepared from lepromas was injected into mice once or several times at weekly intervals by subcutaneous or intravenous route, or by both routes. Animals. were killed at various intervals 2-16 months after injection. At necropsy, lesions were sought by gross inspection. Portions of various organs were removed and ground in mortar to make the homogenates. Smears made from the homogenate were stained by Ziehl-Neelsen's method and examined microscopically. The homogenate treated with one percent sodium hydroxide solution and then inoculated onto the egg yolk medium (for M. lepraemurium; also for M. leprae (?)) and Ogawa 1% egg medium (for cultivable mycobacreria). The tubes were incubated at 37°C for over 3 months. The details of single inoculation experiments and multiple inoculation experiments were shown in Table 2 and 3.Ten experiments containing four with single inoculation and the other six with multiple inoculation were carried out. But one experiment, Expt. (4), exhibited a probable contamination and its results will be described in the following paper separately.In nine experiments, gross findings were all negative. Cultivation trials showed a few, smooth, and buff colonies, supposedly atypical mycobacseria, from two specimens only, but no colonies of mycobacteria, especially suspected of M. leprae, have been isolated.On the other hand, microscopic examination revealed the presence of acid-fast bacilli in the tissues of various organs. Among the two experiments, Expts. (3)-1 & -2, showed remaykable microscopscal findings were summasized in Table 6. As shown, in the subcutaneous experiment Expt. (3)-1, acid-fast bacilli found in the injection site and superficial lymph nodes, and none of the tissue of viscera. In the intravenous experiment, Expt. (3)-2, acid-fast bacilli were detected in the spleen, liver and lungs, but smaller in number, comparing with those of the injection site just mentioned. No bacilli were found in the superficial lymph nodes. In both of the experiments the acidfast bacilli had a tendency to decrease in number steadily. In where numbers of bacilli were present, globi were often seen, but these were usually small in size and loose in arrangement. And, indeed, it was uncertain whether the bacilli had multiplied within the tissue or not. The microscopic findings obtained in all the experiments were summarized in Table 7.As the materials of leproma used differed from experiment to experiment, it was impossible to compare directly the values of percentage for smear-positive specimens. As a whole, however, it seemed fairly justified in concluding that the microscopic findings were superior in the multiple inoculation than in the single inoculation. This fact was accorded with the observations reported by previous workers.ACKNOWLEDGMENTThis work was supported in part by grants from U. S.-Japan Cooperative Medical Science Program.

Tuberculosis of the spermatic cord. Case report.

A 66-year-old man was operated upon for a solid tumour in the spermatic cord. The tumour consisted of epithelioid cell granulomatous inflammation. Tubercle bacilli could not be seen in specimens stained by the Ziehl-Neelsen method but could be demonstrated by fluorescence microscopy. The patient had been operated upon twice earlier, 8 and 4 years ago, for hypertrophy of the prostate. Reexamination of specimens taken from his prostate on these occasions imply that even then he had tuberculosis of the prostate. The ductus deferens was free from inflammatory changes. It is likely that spread of infection from the prostate had occurred via the lymphatics.

Studies on murine leprosy bacillus. XII. Reproduction of the disease in mice using small doses of the thirteenth subculture of the supposed Hawaiian strain of Mycobacterium lepracemurium

As reported previously, an attemp to produce the disease in mice with the supposed Hawaiian strain was repeated four times and each of the tests gave positive results. In the present study, with an aim to know a minimum infecting dose, similar animal experiment was performed by the use of much smaller doses of the bacilli.The animals, ddN strain, were injected with three different doses of the 13th subculture (10-4, 10-5 and 10-6mg of bacilli) either subcutaneously or intravenously, and killed at intervals between 7 to 11.5 months after inoculation (Table 1). The macroscopic apperance of the organs was noted ; smears of spleen, liver, luns, kidneys, superficial lymph nodes and local lesion were stained by Ziehl-Neelsen's method and rated for the numbers of acid-fast bacilli as well as for the numbers of globi. In some instances histopathologic examination was also performed. All of the specimens removed were sumbitted to cultural recovery.The experiment showed that the injection of the bacillus in each dose, whether subcutaneously or intravenously administered, produced the disease, which was characterized by such positive findings as macroscopic and microscopic involvement, increase in acidfast bacilli, appearence of globi, and recovery on culture of the tissues (Tables 2, 3, 4, 5 and 6). The characteristics of the bacilli reisolated were almost the same as those of the bacilli injected originally.It must be conceded that reproduction of the disease was successful even with the amallest dose (10-6mg) of the bacilli. The infection was slowly progressive and the extent of disease was more pronounced in animals inoculated intravenously than in those subcutaneously. Finally a word must be said about the decontaminating agent. In this study 4% sodium hydroxide was used, for the first time, for treating some of the specimens. The result in dicates that this stronger decontamination method is also available for the isolation of murine leprosy bacilli from pathologic materials (Table 7).

Effect of prior oxidation on the acid-fastness of mycobacteria.

Oxidants which attack ethylenes to form aldehydes were effective in increasing the acid-fastness of mycobacteria. Potassium permanganate, performic acid, and peracetic acid were found to be effective. With the permanganate Ziehl-Neelsen stain, acid-fast bacilli seemed to be more strongly stained and more numerous than they did with the unmodified Ziehl-Neelsen method. Moreover, as a practical advantage, the bacilli could be discerned at lower magnification.

Acridine orange as a fluorescent counterstain with the auramine acid-fast stain

Evidence is presented to show that acridine orange is an effective counterstain when used with the auramine-O acid-fast stain. The non-acid-fast material is stained orange to red and the acid-fast bacilli fluoresce yellow to yellow-green.When 133 sputum specimens were examined by the new method and by other fluorescence acid-fast methods, no significant difference in the number of acid-fast positive smears was found. Furthermore, more acid-fast positive smears were found by fluorescence acid-fast microscopy than by the Ziehl-Neelsen method. Culture on Löwenstein-Jensen medium was used as a control on 100 of the specimens.