Negative Staining Technique Research Articles

Transhepatic arterial approaches for ICG injection to guide laparoscopic anatomical hepatectomy: A case series study

Laparoscopic anatomical hepatectomy guided by near-infrared fluorescence imaging (NIR-FI) has been utilized extensively. However, it is difficult to resect "cone units" above the third branch of the Glissonean pedicle in the right posterior lobe using the laparoscopic positive or negative staining techniques. Therefore, we undertook a new laparoscopic segmentectomy based on the concept of "cone unit" assisted by interventional radiology combined with NIR-FI. Laparoscopic segmentectomy guided by NIR-FI via super-selective hepatic arteriography and trans-arterial injection of ICG was carried out on 13 patients with early-stage HCC between September 2020 and January 2022.11 of cases were successful, and relevant pathological characteristics and perioperative outcomes were retrospectively analyzed. Two cases failed NIR-FI out of which one case involved over-staining to the non-target segment, and in the other case, which was to undergo laparoscopic segment V resection, only the ventral segment was stained while the imaging of the dorsal segment failed. In the intraoperative conditions, the tumor safe margin was 1.1 (0.7-1.55) cm, the interventional operation time was 50 (45.5-60.5) minutes, the operation time was 280 (242.5-307.5) minutes, the blood loss was 100 (50-200) ml, the postoperative hospital stay was 5 (4.5-5.5) days. No cases converted to laparotomy, and no serious postoperative complications developed. NIR-FI through super-selective hepatic arteriography and trans-arterial injection of ICG can provide a clear and lasting navigation aid for laparoscopic segmentectomy, which may have positive implication for early-stage HCC with poor preoperative liver reserves.

Fluorescent Laparoscopic Central Hepatectomy for Liver Cancer Using Indocyanine Green Negative Staining.

Laparoscopic hepatectomy is an important treatment method for liver cancer. In the past, the resection boundary was usually determined by intraoperative ultrasound, important vascular structures, and surgeon experience. With the development of anatomical hepatectomy, visual surgery technology has gradually been applied to this type of surgery, particularly indocyanine green (ICG)-guided anatomical hepatectomy. As ICG can be specifically ingested by hepatocytes and used for fluorescence tracing, negative staining techniques have been applied according to different tumor positions. Under ICG fluorescent guidance, the surface boundary and deep resection plane can be more accurately displayed during liver resection. Thus, the tumor-bearing liver segment can be anatomically removed, which helps to avoid damage to important vessels and reduce ischemia or congestion of the remaining liver tissue. Finally, the incidence of postoperative biliary fistula and liver dysfunction is reduced; therefore, a better prognosis is obtained after the resection of liver cancer. Centrally located liver cancer is usually defined as a tumor located at segments 4, 5, or 8 that requires resection of the middle section of the liver. These are among the most difficult hepatectomies to perform because of the large surgical wounds and multiple vessel transections. Based on the specific tumor location, we formulated the required resection ranges by designing personalized fluorescent staining strategies. By completing anatomical resection based on the portal territory, this work aims to achieve the best therapeutic effect.

An Overview of Laboratory Diagnosis of Central Nervous System Viral Infections

Central nervous system (CNS) infection is a serious illness that can lead to death. CNS infections include meningitis, encephalitis, brain abscesses and myelitis. These diseases are caused by causative agents like bacteria, fungi, parasites, and protozoa, but most commonly by viral infections. To combat this issue, accurate diagnosis of etiological agents at an early stage is crucial for appropriate treatment, control of the disease and prevent from becoming life-threatening to the patients. This review paper summarises the main laboratory diagnostic methods for CNS infections caused by viruses ranging from conventional to molecular methods. Conventional isolation methods are considered the ‘gold standard’ as they provide accurate evidence, but require highly skilled personnel, are time-consuming, critical in cell type selection and are useless for non-cultivable viruses. Electron microscopy allows recognition of viral morphology and ultrastructural details as the principle of virus identification through negative staining or thin section technique (suitable for tissue or cell specimens). However, it offers low sensitivity and requires at least 106 virions per millilitre or milligram in the specimen to be detectable by microscopy. Immunological-based methods have been extensively applied for viral diagnosis by detecting the antiviral antibodies or viral antigens in clinical samples. While these methods provided high sensitivity and specificity, the incubation and window period of an infection may give false-negative results. Lastly, molecular detections have many advantages such as high sensitivity, specificity, rapid, require a small amount of sample, simultaneous detection of multiple different viruses, and produce both qualitative and quantitative results.

Low occurrence of Cryptosporidium canis in feces of dogs seroreactive for Leishmania spp.

In this study, we investigated the occurrence and characterized molecularly Cryptosporidium oocysts in feces of dogs seroreactive for Leishmania spp. We hypothesized that the clinical staging of canine visceral leishmaniasis (CVL) influences the occurrence and intensity of Cryptosporidium spp. oocyst shedding in feces from the canine host due to the immunosuppression induced by Leishmania infantum. Fecal samples from 101 dogs with anti-Leishmania spp. antibodies detected by Dual-Path Platform (DPP®) immunochromatographic test (Biomanguinhos) and/or enzyme-linked immunosorbent assay (Biomanguinhos) were concentrated by the centrifuge-sedimentation method in water-ethyl acetate. The sediments were submitted to the technique of negative staining with malachite green and observed under an optical microscope (400× magnification). The occurrence was 0.99% (95 confidence interval 0.00%- 2.93%). A single Cryptosporidium oocyst was found in one of the samples examined by microscopy, and the result was confirmed by nested-PCR. The amplicon sequence showed 100% genetic identity with Cryptosporidium canis. The dog presented the following clinical signs suggestive of CVL: cachexia, generalized alopecia, pale colored gingival mucosa, splenomegaly, and onychogryphosis. We conclude that there is no evidence that the oligosymptomatic and symptomatic clinical staging of dogs seroreactive for Leishmania spp. influenced the occurrence and intensity of C. canis oocyst shedding in the canine host.

Outbreak of paramyxovirus in Chestnut-bellied Seed-Finch (Sporophila angolensis). Transmission electron microscopy diagnosis / Surto de paramixovirose em curiós Sporophila angolensis). Diagnóstico por microscopia eletrônica de transmissão

The Chestnut-bellied Seed-Finch (Sporophila angolensis) is a bird constantly sought after as a cage bird, which is the main threat and the cause of its disappearance from the most inhabited regions of the country. Paramyxovirus, present in most breeding sites and Ecological Parks, is the disease that most affects birds with a high mortality rate, causing significant losses, both economic and to nature. Avian paramyxoviruses belong to the family Paramyxoviridae, which includes 21 serotypes (APMVs-1-21). Serotypes 2 and 3 are the ones that most infect passerines. Type 2 causes respiratory disorders, while the disease caused by serotype 3 is characterized by pancreatitis, conjunctivitis, vomiting, diarrhea, dyspnea, dysphagia, pancreatitis, CNS symptoms, and death. Free-ranging birds can act as viral reservoirs and spread the disease when subjected to stress factors. During the period of 2009, during the illegal commercialization of Brazilian birds, 218 Chestnut-bellied Seed-Finch were apprehended and sent to the CRAS (Wild Animals Recovery Center) of the Tietê Ecological Park. Of these, 112 arrived dead and the rest died during the year. About 35 birds were sent to the Electron Microscopy Laboratory of the Biological institute, for research on viral agents. During the necropsy, it was observed that the intestines were dilated, containing yellowish and watery stools. Small intestine fragments were processed by negative staining (rapid preparation), antigen immunolabeling with colloidal gold particles and resin embedding. Under the transmission electron microscope, using the negative staining technique, pleomorphic, rounded or elongated, enveloped paramyxovirus particles containing helical herring-bone like nucleocapsid, measuring between 100 and 500 nm in diameter, were visualized in the samples of all birds. The antigen-antibody reaction was enhanced by colloidal gold particles. Ultrathin sections of the small intestine revealed the presence of nuclei with marginalized chromatin, containing intranuclear inclusions; amorphous intracytoplasmic inclusions, formed by helical nucleocapsids, complete particles measuring from 100 to 250 nm and incomplete ones, budding from the plasma membrane.

Detection of avipoxvirus in a cutaneous lesion of a swallow tanager (Tersina viridis) by transmission electron microscopy / Detecção de avipoxvírus em lesões cutâneas de saí-andorinha (Tersina viridis) por microscopia eletrônica de transmissão

Avianpox is a highly contagious disease that affects worldwide both commercial and wild birds. The etiologic agent is a poxvirus, belonging to the Poxviridae family and Avipoxvirus genus. The disease manifests itself in three ways, cutaneous, diphtheric and septicemic. In June 2007, during the illegal commercialization of birds, a Swallow tanager (Tersina viridis) was apprehended by the Forestry Police, among other birds, which was sent to the Tietê Ecological Park, São Paulo, SP, Brazil. After one month, the bird presented skin lesions on its legs, anorexia, emaciation, mobility difficulties, diarrhea, dehydration and death, and was sent to the Electron Microscopy Laboratory of the Biological Institute, São Paulo, SP, Brazil, to search for viral agents. Fragments of skin lesions and crusts were collected and processed for transmission electron microscopy using the techniques of negative staining (rapid preparation) and immunocytochemistry (immunollabelling with colloidal gold particles). Under the transmission electron microscope using the negative staining technique, a large number of typical poxvirus particles ovoid in shape, showing irregular arrangement of the tubules on the outer membrane, some enveloped, measuring an average of 240 nm in length x 200 nm in diameter, were seen in suspension of skin lesions or crusts. . In the immunocytochemistry technique the antigen-antibody reaction was strongly enhanced by the dense particles of colloidal gold on poxviruses.

Laparoscopic anatomical liver resection for malignancies using positive or negative staining technique with intraoperative indocyanine green-fluorescence imaging.

Indications for a minimally invasive resections are increasing worldwide, but respecting anatomical planes during intraparenchymal transection is demanding. Intraoperative ICG fluorescence staining of liver parenchyma has been introduced as a tool for real-time intraoperative guidance. The aim of this study is to make a systematic review of the current relevant literature on indications, techniques, and results of laparoscopic anatomical liver resection (LALR) using intraoperative indocyanine green (ICG) fluorescence for positive and negative staining of liver segments in patients affected by liver malignancies. Electronic bibliographical databases (MEDLINE and PubMed) were searched according to the PRISMA criteria. English language articles meeting the selection criteria and published until June 2020 were retrieved and reviewed. a total of 86 articles were initially found and 11 articles were finally included in the analysis with a total of 83 patients treated. Sixty-two patients (74.6%) underwent mono-segmentectomies. Thirty-five patients (42.1%) underwent the positive staining technique, and forty-eight patients (57.8%) the negative staining technique. The positive or negative indocyanine green staining technique with real-time fluorescence guidance is an emerging and promising approach. However, the technique has to be standardized and advantages in terms of oncologic results still need validation in further studies.

Intraoperative guidance using ICG fluorescence imaging system for safe and precise laparoscopic liver resection.

Laparoscopic liver resection (LLR) has been spread as minimally invasive surgery for liver disease. Advances in surgical technique and devices enabled us to perform various procedures of LLR. Indocyanine green (ICG) fluorescence imaging has been suggested as useful tool to identify liver tumors, anatomical territory of liver parenchyma, and cholangiography in open liver surgery. Due to recent development, this technology can be applied in LLR. we describe safe and effective using of the ICG fluorescence imaging during LLR. From September 2013 to August 2019, 34 patients were performed LLR using a total of 46 procedures by ICG fluorescence imaging system for purposes including identification of anatomic domain of the liver in 12 LLRs, detection of liver tumors in 30 nodules, or intraoperative cholangiography in 4 LLRs. During the detection of liver tumors, 25 nodules in 30 malignant to benign tumors were positively detected (83.3%). Although there has been no publication regarding information on ICG fluorescence imaging of low grade malignant or benign tumors, we found positive emission in focal nodular hyperplasia, an angiomyolipoma, and an intraductal papillary neoplasm of the bile duct. The identification of anatomic domain in the liver was successful in all 12 LLRs with negative and positive staining techniques. In the intraoperative cholangiography, all 4 tests were successfully performed. One of 4 patients were found to have biliary leakage which was repaired intraoperatively. The ICG fluorescence imaging could be useful in safe and precise performance of LLR.

‘Seven shades of Cryptosporidium’

In this paper, we describe a case of clinically unsuspected cryptosporidiosis that was diagnosed through systematic screening of coccidia using the little known, but highly reliable, extemporaneous Heine technique. This is significant because this zoonosis is largely underestimated, probably since most of the laboratories may not look for Cryptosporidium unless specifically requested. Moreover, the performance of the modified Ziehl-Neelsen staining, still considered as "gold standard", remains variable owing to the increasing proportion of non-acid-fast oocysts "ghosts" in resolving infections. Contrastingly, the negative staining technique of Heine highlights oocysts irrespectively of the time since diarrhoea onset. In addition, we provide a rare panel of fluorescent images that would be useful for microscopists.

Preparation of dimethylaminopropyl octadecanamide/stearic acid vesicles incorporating azobenzene and their UV-responsive release property

UV-responsive vesicle was prepared by incorporating azobenzene in the vesicular membrane composed of dimethylaminopropyl octadecanamide (DMAPODA) and stearic acid (SA). Azobenzene intercalated in the vesicular membrane underwent cis-to-trans isomerization under the UV irradiation, confirmed by UV spectroscopy. The phase transition temperature of vesicle was found to be around 51.2 °C by differential scanning calorimetry. Using a negative staining technique, the vesicles were found as circular and irregular-shaped particles on the TEM photo. The mean hydrodynamic diameter of vesicles increased from 199.4 to 1597.3 nm when the lipid/azobenzene ratio increased from 100/0 to 100/5. The zeta potential of DMAPODA/SA vesicle decreased from about + 60 mV to – 60 mV when the pH value of medium increased from 3.0 to 10.0, and the inclusion of azobenzene in the vesicular membrane had little effect on the zeta potential. UV irradiation markedly promoted the release of carboxylic fluorescein loaded in vesicle incorporating azobenzene, possibly because the cis-to-trans photoisomerization would disturb the well-ordered crystalline structure of the vesicular membrane.

Indocyanine green fluorescence-guided laparoscopic left hemihepatectomy: efficacy of negative staining for precise parenchymal transection and bile duct resection

Purpose: ICG fluorescence imaging is useful to locate segmental boundaries for anatomical resection and to delineate biliary anatomy. Its application in laparoscopic liver surgery is being recently explored and perceived of usefulness given the lack of tactile sensation. This video documents the application of the negative ICG staining technique for an efficient fluorescence guide in pure laparoscopic left hemihepatectomy, for both precise parenchymal transection and bile duct resection. Method: The patient is affected by solitary intrahepatic recurrence of colorectal liver metastasis between Sg 3 and 4 with endobiliary thrombus dilating the left hemiliver biliary tree. The hilar approach is used to isolate the left hepatic artery and ipsilateral portal branch, and the inflow for the left hemiliver is selectively clamped. A solution of 10 mg of ICG diluted in 10 mL saline solution is administered i.v. and the camera switched to the near infrared light. Results: After 2 mins the right hemiliver becomes fluorescent and left hemiliver counterstained on the glissonian surface. The proper transection line is marked, and the transection started. Differently from the traditional vascular demarcation, the ICG fluorescence boundary remains highly visible along the deep transection planes which allows to follow a clear and clean dissection plane during all the transection time. The transection is continued up to the hilar plate until the left portal branch and ipsilateral hepatic artery are encountered, which are both secured with clips before bulldog removal it is ensured that the left hemiliver remains unperfused by ICG, and the two vessels are then interrupted. The left bile duct is identified intraparenchymally, with the endobiliary thrombus inside remaining counterstained, and it is encircled distally to its endobiliary bulging thus allowing precise bile duct section with sufficient free margin at first frozen section. The transection is carried forward up to the identification of the left hepatic vein, which is sectioned by vascular stapler, and the specimen then extracted after biliostatic and hemostatic check. Conclusion: ICG fluorescence imaging with negative staining is useful not only for segmental anatomical resections but safe and efficient technique for both precise parenchymal transection and bile duct resection during anatomical major hemihepatectomy.

Utility of ICG fluorescence imaging with vessel clamp for ileocecal resection while preserving ileal conduit constructed after previous total cystectomy

BackgroundIndocyanine green (ICG) is useful for evaluating the intestinal perfusion of anastomosis. Especially for patients with prior surgeries, ICG imaging enables surgeons in visualizing the anatomical field. Here, we reported the positive and negative staining techniques of ICG fluorescence with vessel clamp for determining the optimal resection area of vessels and mesentery.Case presentationAn 80-year-old man, who had an ileal conduit constructed after a prior total cystectomy, was diagnosed with ascending colon cancer. Although the tumor-feeding vessel was primarily the ileocecal artery, there was no detailed information about the blood running through the ileal conduit. At first, the ascending colon and the marginal vessels were transected at distal side of the tumor. Next, both, the ileocecal artery and the marginal artery of oral side of the ileal anastomotic site were clamped. Finally, we injected ICG intravenously to assess the blood flow. As a result, the blood flow between the ileal anastomotic site and transected ascending colon was not identified (negative staining). Therefore, we cut the root of the ileocecal artery, and dissected the peripheral mesocolon including the ileal anastomotic site. After the ileo-ascending colon anastomosis, we injected ICG intravenously again. The blood flow to the ileal conduit was preserved (positive staining).ConclusionICG fluorescence imaging with vessel clamp can clearly visualize the demarcation line between ischemic and non-ischemic intestinal tract. In colorectal surgeries, this technique is useful to assess the anastomotic perfusion and determine optimal dissection area of vessels and mesentery in secondary intestinal surgery.

Outbreak of Encephalitis Caused by Human Herpesvirus Type 1 (HSV-1) in NonHuman Captive Primates in São Paulo, SP, Brazil

Human herpesvirus type 1 (HSV-1), a Alphaherpesvirinae, has a broad range of cross-species infectivity with considerable variation in expression and disease severity among different hosts. Infection in humans, results in mild disease characterized by mucocutaneous lesions. In some species of nonhuman primates, however, the infection can be lethal. Transmission occurs through food contaminated by humans with labial herpes, offered to monkeys, constituting an important anthropozoonosis. In this study, we describe the occurrence of two outbreaks of encephalitis caused by HSV-1, occurring in the State of São Paulo, Brazil. The first outbreak affected a Brown howler monkey and three marmosets. The Brown howler monkey had tongue ulcers and enteritis and the marmosets were found dead without presenting clinical symptomatology. The second outbreak affected ten marmosets that were found dead suddenly in the enclosure without presenting symptoms or clinical signs. Organ fragments were processed for transmission electron microscopy, histopathology and inoculation in cell culture techniques. Ballooning degeneration, foci of monolymphocytic inflammatory reaction, corpuscle of eosinophilic inclusion, monolymphocytic meningitis and desquamative monolymphocytic enterocolitis, were the main lesions observed by histopathology. By the negative staining technique, enveloped and non-enveloped, herpesvirus particles were found, measuring 120-200 nm in diameter in all samples of organ fragments. The presence of aggregates formed by antigenantibody complex, characterized the positive result obtained in the immunoelectron microscopy technique for HSV-1. Using the immunocytochemistry technique, the antigen-antibody interaction was strongly enhanced by the colloidal gold particles over the herpesvirus, confirming the presence of HSV-1. Intranuclear incomplete particles measuring 80-100 nm in diameter and complete or enveloped scattered in the cytoplasm, were visualized in ultrathin sections of liver and cell culture measuring 140 nm of diameter. Immature particles budding from cell membranes were also observed. In viral isolation in VERO cells, typical cytopathic effect was observed in brain samples.

Comparison of epiretinal membrane peel after dual staining versus en bloc peel using negative staining technique

Purpose: To compare the completeness of ILM peel in cases of Idiopathic Epiretinal Membranes following double staining and double peeling versus en bloc dissection of both using negative staining method.
 Study Design: Quasi experimental study.
 Place and Duration of Study: Jinnah Hospital Lahore, from October 2017 to March 2018.
 Material and Methods: Forty eyes of forty patients with Idiopathic Epiretinal membranes (ERM) were selected by non-probability convenient method and divided into two groups. In group A, the ERM was initially stained with Trypan blue and then peeled. Subsequently the macula was re-stained with Brilliant blue G (BBG). In group B, ERM and ILM were successfully peeled together as a single membrane utilizing the single block technique, after a desired negative staining effect was obtained by injecting BBG. The thoroughness of the ILM peel was established using another injection of BBG although a few remains were often identified outside the central macular area, no other dyes were used.
 Results: In group A, 8 of the 20 eyes (40%) had full undisturbed ILM present whereas in 10 eyes (50%) the ILM was there but damaged, only 2 eyes (10%) had near total ILM removed. In group B, the dual peeling was successful in 17 of the 20 cases (85%). There has been no recurrence of ERM until now.
 Conclusion: Instead of peeling twice, the single en bloc negative staining technique with Brilliant blue G is a safer and more effective method for removal of ERM together with ILM.

Methods for particle size reduction of liposomal amphotericin B

Liposomal amphotericin B were prepared by thin film hydration technique. Particle size was reduced by ultrasonic device and high-pressure homogeneous device. The size distribution was determined by dynamic light scattering by the Zetasizer ZS90 equipment. The morphology of amphotericin B liposomes was observed by Transmission electronic microscopy (TEM) with negative staining technique. The result shows that combining high-pressure homogenization and membrane extrusion provided monolayer liposomal amphotericin B with the particle size less than 200 nm and homogeneously (PDI < 0.3).

Rapid Viral Diagnosis of Orthopoxviruses by Electron Microscopy: Optional or a Must?

Diagnostic electron microscopy (DEM) was an essential component of viral diagnosis until the development of highly sensitive nucleic acid amplification techniques (NAT). The simple negative staining technique of DEM was applied widely to smallpox diagnosis until the world-wide eradication of the human-specific pathogen in 1980. Since then, the threat of smallpox re-emerging through laboratory escape, molecular manipulation, synthetic biology or bioterrorism has not totally disappeared and would be a major problem in an unvaccinated population. Other animal poxviruses may also emerge as human pathogens. With its rapid results (only a few minutes after arrival of the specimen), no requirement for specific reagents and its “open view”, DEM remains an important component of virus diagnosis, particularly because it can easily and reliably distinguish smallpox virus or any other member of the orthopoxvirus (OPV) genus from parapoxviruses (PPV) and the far more common and less serious herpesviruses (herpes simplex and varicella zoster). Preparation, enrichment, examination, internal standards and suitable organisations are discussed to make clear its continuing value as a diagnostic technique.

Detection of Parapoxvirus in goats during contagious ecthyma outbreak in Ceará State, Brazil by transmission electron microscopy techniques

Contagious ecthyma or contagious pustular dermatitis, is a viral skin disease that occurs in sheep, goats and wild ruminants and is characterized by the formation of papules, nodules or vesicles that progress into thick crusts or heavy scabs on the lips, gingiva and tongue, caused by a member of the Parapoxvirus genus. Humans are occasionally affected constituting important zoonosis. The disease not only has an economic impact on farmers worldwide but also has a considerable negative effect on animal welfare. In this study, a contagious ecthyma outbreak which occurred in one flock with 90 goats located in the Ceara State, Brazil, was described. Twenty-two goats older than 6 months were affected. The animals presented crusted lesions on the buccal region, tongue, udder and teats, which began with swelling in the mouth area. Dried crusts and serum collected were processed for transmission electron microscopy utilizing, negative staining (rapid preparation), Immunocytochemistry (immunolabelling with colloidal gold particles) and resin embedding techniques. At the Philips EM 208 transmission electron microscopy all the samples were analyzed by negative staining technique and a great number of parapoxvirus particles ovoid or cylindrical, showing two morphological forms, a mulberry (M) form with a distinctive crisscross filament pattern derived from the superimposition of upper and lower virion surfaces and a capsular (C) form caused by stain penetration and distention of the virion core, measuring 300 x 180 nm was observed. Antigen antibody reaction was increased by the colloidal gold particles. In the ultrathin sections of crusts, we verified the presence of three types of intracytoplasmic inclusion bodies, type A or Bollinger inclusion bodies, outlined by membrane, presented in it is interior, oval, mature or complete viral particles, measuring on the average, 225nm x 130 nm, showing an inner dumbell-shaped core, two lateral bodies and an external envelope, or cigar shaped core. In the type B electron dense inclusions bodies, were visualized parapoxvirus particles budding of dense and amorphous material. Fibrillar intracytoplasmic inclusions were also found located between the virions, consisting of groups of fibrils, arranged in groups or concentrically in the middle of the granular material. Intracytoplasmic vesicles outlined by membranes, measuring 560 x 420 nm, containing granular material in its interior were also observed. The nuclei showed an aspect deformed.

Presence of herpesvírus in diseased fishes

Herpesviruses that infect fishes belong to the Herpesvirales order and Alloherpesvirus family. In these species, the different types of herpesvirus can cause tumors, adenocarcinoma and skin lesions. This study aims detect to presence of herpesvirus in fishes from commercial, recreation or experimental creations of the States of Sao Paulo and Minas Gerais, Brazil. Organ fragments and lesions of 53 fish species coming of mortality cases were forwarded at Biological Institute for examination by transmission electron microscopy by research of etiological agent. By transmission electron microscopy through negative staining technique, were observed herpes virus-like particles in 46 fishes and through embedding resin technique, in ultrathin sections were visualized herpes virus immature particles, measuring 90-110nm in diameter, located in the nuclei and complete particles measuring 160nm. In the histopathology technique, lesions associated with the virus as corpuscles inclusion, papillomas, and dermal lesions and in the gills were observed in 27 fishes. The evaluated techniques of TEM and the histopathology were effective for the rapid detection of herpesvirus in the examined samples.

Portal vein territory identification using indocyanine green fluorescence imaging: Technical details and short-term outcomes.

Portal vein (PV) territory identification during liver resection may be performed using indocyanine green (ICG) fluorescence imaging technique. However, the technical details of the fluorescence staining technique have not been fully elucidated. This study was performed to demonstrate the technical details of PV territory identification using fluorescence imaging and evaluates the short-term outcomes. From 2011 to 2015, 105 underwent liver resection at the University of Tokyo Hospital with one of the following fluorescence staining techniques by transhepatic PV injection or intravenous injection of ICG: single staining (n = 36), multiple staining (n = 31), counterstaining (n = 22), negative staining (n = 13), or paradoxical negative staining (n = 3). The PV territory was identified as a region with fluorescence or a defect of fluorescence using one of the five staining techniques. ICG was administered by transhepatic PV injection in all but the negative staining technique, which employed intravenous injection. No adverse events associated with the ICG administration occurred. The mortality, postoperative total morbidity, and the major complication (Clavien-Dindo grade ≥III) rates were 0.0%, 14.3%, and 7.6%. We have demonstrated the technical details of five types of fluorescence staining techniques. These techniques are safe to perform and facilitate clear visualization of the PV territory in real time, enhancing the efficacy of anatomical removal of such territories.

Characterization of the chemical structures and physical properties of exopolysaccharides produced by various Streptococcus thermophilus strains

Exopolysaccharides (EPS) produced by some lactic acid bacteria are often used by the dairy industry to improve the rheological and physical properties of yogurt, but the relationship between their structure and functional effect is still unclear. The EPS from different species, or different strains from the same species, may differ in terms of molar mass, repeating unit structure, and EPS yield during fermentation of milk. This study aimed to characterize the detailed properties of EPS produced from 7 strains of Streptococcus thermophilus, which is one of the key cultures used for yogurt manufacture. Milk was fermented with strains DGCC 7698, DGCC 7710, DGCC 7785, ST-10255y, St-143, STCth-9204, and ST4239. These strains were selected because they have been used in previous studies on yogurt texture, but a complete description of their EPS structural properties has not yet been reported. All strains were fermented under a similar acidification rate by adjusting the level of supplementation with peptone or the inoculation level, which allowed for a comparison of EPS yields under similar growth conditions (reconstituted skim milk at 40°C). The EPS from each strain was isolated and the weight-average molar mass and z-average root mean square radius determined using size-exclusion chromatography multiangle laser light scattering. The monosaccharide composition of EPS was determined using gas chromatography-mass spectrometry, and repeating unit structure was determined using nuclear magnetic resonance spectroscopy. The weight-average molar mass values of EPS ranged from 0.14 to 1.61 × 106 g/mol. All 7 EPS samples were uncharged. The strains ST-10255y and ST4239 had EPS with the same repeating unit structure. The monosaccharide compositions of the various EPS were mainly composed of glucose and galactose, with low levels of rhamnose in the EPS isolated from DGCC 7698, and N-acetylgalactosamine in the EPS from DGCC 7785, ST-10255y, and ST4239. The yields of EPS (measured when fermented milks reached pH 4.6) ranged from 8.0 to 76.4 mg of glucose equivalents/kg. In addition to (free) EPS, some strains were also able to produce capsular polysaccharide (associated with the bacterial cells) when observed with negative staining technique. The results of our study will help the dairy industry to better understand the mechanism by which different strains of Streptococcus thermophilus affect yogurt texture.