Methodological Proposal for the Adequate Use of the Osteotechnics Technique / Propuesta Metodológica para el uso Adecuado de la Técnica Osteotécnica

SUMMARY: Osteotechnics is one of the different anatomical preservation techniques and can be defined as the technique designed to prepare, clean, obtain and preserve bone structures that can be used in the teaching, museographic or research field. The osteotechnical technique procedure consists of the following phases: debulk and disjoint, maceration, cooking, cleaning, degreasing, bleaching, and labeling to obtain bone material. Seven phases will be explained in detail, as well as the materials, instruments, quantities of the substances used, and the time required to obtain human bone material. We consider that this article can serve as a guide, given that all the experimentation was carried out with human biological material. This methodological proposal could be consolidated and established based on the experience acquired during the creation of the contemporary skeletal collection of the department of innovation in human biological material (DIMBIH). Therefore, the purpose of our proposal is to provide tools that facilitate the work of those who carry out this work and fundamentally to avoid irreversible or irreparable damage to the osteological material, since it is of great value and difficult to acquire for disciplines as anatomy, veterinary, physical and forensic anthropology, medicine, dentistry and biology.

La osteotecnia es una de las técnicas diferentes de conservación anatómica y puede definirse como la técnica destinada a preparar, limpiar, obtener y conservar estructuras óseas que pueden ser utilizadas en el ámbito docente, museográfico o de investigación. El procedimiento de la técnica osteotécnica consta de las siguientes fases: descarnado y desarticulado, maceración, cocción, limpieza, desengrase, blanqueo y marcaje para la obtención de material óseo. Se explicarán en detalle siete fases, así como los materiales, instrumentos, cantidades de las sustancias utilizadas y el tiempo necesario para obtener material óseo humano. Consideramos que este artículo puede servir de guía, dado que toda la experimentación se realizó con material biológico humano. Esta propuesta metodológica pudo consolidarse y establecerse a partir de la experiencia adquirida durante la creación de la colección esquelética contemporánea del Departamento de Innovación en Material Biológico Humano (DIMBIH). Por lo tanto, el propósito de nuestra propuesta es brindar herramientas que faciliten el trabajo de quienes realizan este trabajo y fundamentalmente evitar daños irreversibles o irreparables en el material osteológico, ya que es de gran valor y difícil adquisición para las disciplinas como la anatomía, veterinaria, antropología física y forense, medicina, odontología y biología.

Development of a new protocol for freeze-drying preservation of Pseudoalteromonas nigrifaciens and its protective effect on other marine bacteria

Background: Freeze-drying is known as one of the best methods to preserve bacterial strains. Protectant is the key factor affecting the survival rate of freeze-dried strains. In addition, salinity, bacterial suspension concentration, drying time, and other factors can also affect the survival rate of strains to varying degrees. At present, there are relatively few studies on freeze-drying preservation of marine bacteria. In the present study, we performed the freeze-drying protectant screening and optimized the preservation conditions for Pseudoalteromonas nigrifaciens, which is widely distributed in marine environment. The protective effects of the screened protectants were verified by 18 other marine bacterial strains. Results: The results indicated that the combination of 5.0% (w/v) lactose, 5.0% (w/v) mannitol, 5.0% (w/v) trehalose, 10.0% (w/v) skim milk powder, 0.5% (w/v) ascorbic acid and 0.5% (w/v) gelatin was the best choice for the preservation of P. nigrifaciens. The suggested salinity and concentration of initial cell suspension were 10 g/L NaCl and 1.0 × 109 CFU/mL, respectively. Furthermore, stationary-phase cells were the best choice for the freeze-drying process. The highest survival rate of P. nigrifaciens reached 52.8% when using 5­10% (w/v) skim milk as rehydration medium. Moreover, the other 18 marine strains belonging to Pseudoalteromonas, Vibrio, Photobacterium, Planomicrobium, Edwardsiella, Enterococcus, Bacillus, and Saccharomyces were freezedried under the abovementioned conditions. Their survival rates were 2.3­95.1%. Conclusion: Collectively, our results supported that the protectant mixture and parameters were beneficial for lyophilization of marine bacteria

National production of certified reference fungal cultures / Producción nacional de cultivos fúngicos de referencia certificados

Reference fungal cultures (RFCs) are essential for the internal quality control of laboratories. The production of these cultures requires standardized procedures (IRAM 14950:2016 and ISO 17034:2016 standards) carried out by a recognized and accredited laboratory. The aim of this work was to produce RFC in paper disks of autochthonous strains, characterized by two, homogeneous and stable reference methods traceable at species level. RFC were produced using 14 regional species (7 yeasts and 7 filamentous fungi) from the fungal culture collection (DMic). Paper disks were impregnated with a culture suspension, dried and packed. Homogeneity, viability, identity and purity were verified. Short-and long-term stability at different temperatures and storage times were studied. Characterization of each strain allowed to confirm its identity and to ensure its traceability at international level. Produced batches were homogeneous and stable at -20 ±5 °C for 30 months. This method of production was adequate to produce homogeneous and stable RFC with phenotypic and genotypic characteristics correctly defined and internationally traceable. Standardized procedures were developed for the production of certified RFC that could be transferred to other microorganisms. Providing RFC that represent regional strains allows laboratories to produce more reliable results with a favorable impact on medical diagnosis, the environment or the food industry.

Los cultivos microbianos de referencia (CR) son imprescindibles para el control de calidad interno de los laboratorios. Asegurar su producción requiere de procedimientos estandarizados (IRAM 14950:2016 e ISO 17034:2016) realizados en un laboratorio reconocido y acreditado. El objetivo de este estudio fue producir cultivos fúngicos de referencia en discos de papel, a partir de un panel de cultivos autóctonos caracterizados por dos métodos de referencia, trazables a nivel taxonómico de especie, homogéneos y estables. Se produjeron CR de 14 especies circulantes en Argentina (7 de levaduras y 7 de hongos miceliales), depositadas en la colección de hongos de interés médico (DMic). Los discos de papel fueron embebidos con una suspensión del cultivo por producir, secados y envasados. Se verificó la homogeneidad, viabilidad, identidad y pureza de cada lote. Se evaluó la estabilidad a corto y largo plazo a distintas temperaturas y tiempos de almacenamiento. La caracterización de cada CR nos permitió confirmar su identidad y asegurar su trazabilidad a nivel internacional. Los lotes producidos fueron homogéneos y estables durante 30 meses conservados a -20 ±5 °C. Este método resultó adecuado para producir CR homogéneos y estables, con características fenotípicas y genotípicas correctamente definidas y trazables a nivel internacional. Los procedimientos estandarizados desarrollados en este trabajo pueden ser transferidos para producir CR certificados de otros microorganismos. La provisión de CR que represente cepas regionales permite a los laboratorios producir resultados más confiables con un impacto favorable en el diagnóstico médico, los estudios ambientales y la industria alimenticia.

Conceptos fundamentales del protocolo modificado de plastinación a temperatura ambiente con silicona, con posterior pigmentación, y su aplicación para la conservación de placenta humana / Fundamental concepts of the modified room temperature plastination protocol with silicone, with subsequent pigmentation, and its application for the conservation of human placenta

RESUMEN: El auge experimentado en los últimos años en la aplicación de las técnicas anatómicas para la conservación de muestras anatómicas está directamente relacionado con la necesidad de preservación de los escasos especímenes con que cuentan las instituciones universitarias en relación a aumentar el tiempo de utilización del mismo. En este sentido, la plastinación es la técnica anatómica que más se destaca y que permite preservar por tiempo indeterminado, sin toxicidad, las preparaciones anatómicas. Presentamos el protocolo modificado de plastinación a temperatura ambiente con silicona, desarrollado en el Laboratorio de Plastinación y Técnicas Anatómicas de la Universidad de La Frontera, con el objetivo de aplicarla a la conservación de una placenta humana, la cual posteriormente fue pigmentada para otorgarle un aspecto más cercano a lo real.

SUMMARY: The surge experienced in recent years in the application of anatomical techniques for the conservation of anatomical samples is directly related to the need to preserve the few specimens that university institutions have in relation to increase the time of use of the same. In this sense, the plastination is the anatomical technique that stands out and that allows to preserve indefinitely, without toxicity, the anatomical preparations. We present the modified plastination protocol at room temperature with silicone, developed in the Laboratory of Plastination and Anatomical Techniques of the University of La Frontera, with the aim of applying it to the conservation of a human placenta, which was subsequently pigmented to give it an appearance closer to the real.

Methods to preserve potentially toxigenic fungi

Microorganisms are a source of many high-value compounds which are useful to every living being, such as humans, plants and animals. Since the process of isolating and improving a microorganism can be lengthy and expensive, preserving the obtained characteristic is of paramount importance, so the process does not need to be repeated. Fungi are eukaryotic, achlorophyllous, heterotrophic organisms, usually filamentous, absorb their food, can be either macro or microscopic, propagate themselves by means of spores and store glycogen as a source of storage. Fungi, while infesting food, may produce toxic substances such as mycotoxins. The great genetic diversity of the Kingdom Fungi renders the preservation of fungal cultures for many years relevant. Several international reference mycological culture collections are maintained in many countries. The methodologies that are most fit for preserving microorganisms for extended periods are based on lowering the metabolism until it reaches a stage of artificial dormancy . The goal of this study was to analyze three methods for potentially toxigenic fungal conservation (Castellani's, continuous subculture and lyophilization) and to identify the best among them.

Viability and molecular authentication of Coccidioides spp. isolates from the Instituto de Medicina Tropical de São Paulo culture collection, Brazil / Viabilidade e autenticação molecular de isolados de Coccidioides spp. da coleção de culturas do Instituto de Medicina Tropical de São Paulo, Brasil

Coccidioidomycosis is an emerging fungal disease in Brazil; adequate maintenance and authentication of Coccidioides isolates are essential for research into genetic diversity of the environmental organisms, as well as for understanding the human disease. Seventeen Coccidioides isolates maintained under mineral oil since 1975 in the Instituto de Medicina Tropical de São Paulo (IMTSP) culture collection, Brazil, were evaluated with respect to their viability, morphological characteristics and genetic features in order to authenticate these fungal cultures. Only five isolates were viable after almost 30 years, showing typical morphological characteristics, and sequencing analysis using Coi-F and Coi-R primers revealed 99% identity with Coccidioides genera. These five isolates were then preserved in liquid nitrogen and sterile water, and remained viable after two years of storage under these conditions, maintaining the same features.

Coccidioidomicose é uma doença emergente no Brasil; a manutenção adequada e autenticação de isolados de Coccidioides spp são essenciais para a pesquisa em diversidade genética de micro-organismos, bem como para a compreensão da doença em humanos. Dezessete isolados de Coccidioides preservados em óleo mineral desde 1975 na coleção de culturas do Instituto de Medicina Tropical de São Paulo (IMTSP) foram avaliados com relação à viabilidade, características morfológicas e genéticas, com o objetivo de autenticação das culturas fúngicas. Dos 17 isolados, apenas cinco foram viáveis após quase 30 anos mantidos em óleo mineral, apresentando características morfológicas e moleculares típicas do gênero, o sequenciamento utilizando os oligonucleotídeos Coi-F e Coi-R revelou identidade de 99% com isolados de Coccidioides. Estes cinco isolados foram preservados em nitrogênio líquido e água destilada esterilizada, e permaneceram viáveis após dois anos de armazenamento sob estas condições, mantendo as mesmas características.

Applying multiple displacement amplification to DNA typing in the pathological section / 法医学杂志

OBJECTIVE@#To investigate the feasibility of applying multiple displacement amplification (MDA) to DNA typing in forensic pathological section.@*METHODS@#Ninety-eight pieces of pathological sections were prepared in terms of 3 factors as the period of preservation, tissue types and death ages, and randomized into groups by Latin square by double 7-order design. Silicon bead method was used to extract the DNA template. Compared with the PCR amplification performed directly by AmpFlSTR Identifiler kit in the control group, MDA was performed before amplification in the experimental group. Based on the samples from fresh autopsies as the standard genotypes, the number of detection and the detection rate were analyzed and compared between the experimental group and the control group.@*RESULTS@#Between the control group and the experimental group, there was significantly statistical difference regarding the rate of DNA typing in each period of the tissue sections preserved (P<0.01). The detection rate of the 16 loci in the experimental group was more than 95% when the period of the tissue sections were preserved within 360d. There was significant difference in different tissue types (P<0.01). But there was no significant difference in different death ages (P>0.01).@*CONCLUSION@#MDA is efficacious in DNA typing of forensic pathological sections, for it can improve the DNA template quantification through abating the inhibiting factor's concentration of PCR and reducing the rate of allele drop out (ADO). However, the period of the sections preserved and tissue types would affect the results of genotyping by MDA.

Stool sample storage conditions for the preservation of Giardia intestinalis DNA

Stool is chemically complex and the extraction of DNA from stool samples is extremely difficult. Haemoglobin breakdown products, such as bilirubin, bile acids and mineral ions, that are present in the stool samples, can inhibit DNA amplification and cause molecular assays to produce false-negative results. Therefore, stool storage conditions are highly important for the diagnosis of intestinal parasites and other microorganisms through molecular approaches. In the current study, stool samples that were positive for Giardia intestinalis were collected from five different patients. Each sample was stored using one out of six different storage conditions [room temperature (RT), +4ºC, -20ºC, 70% alcohol, 10% formaldehyde or 2.5% potassium dichromate] for DNA extraction procedures at one, two, three and four weeks. A modified QIAamp Stool Mini Kit procedure was used to isolate the DNA from stored samples. After DNA isolation, polymerase chain reaction (PCR) amplification was performed using primers that target the β-giardin gene. A G. intestinalis-specific 384 bp band was obtained from all of the cyst-containing stool samples that were stored at RT, +4ºC and -20ºC and in 70% alcohol and 2.5% potassium dichromate; however, this band was not produced by samples that had been stored in 10% formaldehyde. Moreover, for the stool samples containing trophozoites, the same G. intestinalis-specific band was only obtained from the samples that were stored in 2.5% potassium dichromate for up to one month. As a result, it appears evident that the most suitable storage condition for stool samples to permit the isolation of G. intestinalis DNA is in 2.5% potassium dichromate; under these conditions, stool samples may be stored for one month.

Comparação entre suplementos homólogos do leite humano e um suplemento comercial para recém-nascidos de muito baixo peso / Comparison between homologous human milk supplements and a commercial supplement for very low birth weight infants

OBJETIVOS: Descrever a metodologia de preparo de dois aditivos, líquido e em pó, derivados do leite humano e comparar a constituição com aditivo comercial FM85®. MÉTODOS: Foram utilizadas 40 amostras de leite humano para o preparo dos suplementos líquido e em pó. Ambos passaram por três fases de preparo: desnate, evaporação e retirada da lactose. Após essas fases, o suplemento líquido está pronto, e o em pó necessita da quarta fase - a liofilização. Em cada amostra dos suplementos líquido e em pó, foram adicionados, respectivamente, 80 mL (grupo I) e 100 mL (grupo II) de pool de leite humano de banco. Para comparação, 20 amostras de 100 mL do pool foram acrescidas de 5 g do suplemento FM85® (Nestlé) (grupo III). Realizaram-se análises de hidratos de carbono, proteína, lipídios, cálcio, fósforo, sódio, osmolalidade e conteúdo calórico, considerando diferença significativa p < 0,05. RESULTADOS: Os grupos I, II e III mostraram, respectivamente, os seguintes resultados: proteínas = 1,81, 2,38 e 1,96 g/dL (p < 0,001); hidratos de carbono = 6,70, 7,25 e 10,06 g/dL (p = 0,006); gordura = 3,75, 3,75 e 3,73 g/dL (p = 0,96); cálcio = 36,92, 44,75 e 79,37 mg/dL (p = 0,001); fósforo = 20,02, 23,28 e 56,30 mg/dL (p = 0,02); sódio = 14,32, 14,40 e 20,33 mEq/L (p = 0,143); osmolalidade = 391,45, 412,47 e 431, 00 mOsmol/kgH2O (p = 0,074); e conteúdo calórico = 67,78, 72,27 e 81,65 kcal (p = 0,001). CONCLUSÃO: Os aditivos estudados diferem significativamente do aditivo comercial FM85® em alguns de seus constituintes, e a sua constituição pode ou não atender às quantidades de nutrientes propostas pelas recomendações mais recentes.

OBJECTIVES: To describe the methodology for the preparation of two additives derived from human milk, liquid and powdered, and to compare this composition with the commercial additive FM85®. METHODS: For the preparation of the liquid and powdered supplements, 40 samples of human milk were used. Both supplements have been through three preparation phases: skimming, evaporation and lactose removal. After these phases, the liquid supplement is ready, and the powdered requires a fourth phase - lyophilization. To each sample of the liquid and powdered supplements were added, respectively, 80 mL (group I) and 100 mL (group II) of pooled banked human milk. For comparison, 20 samples of 100 mL of the pool were added to 5 g of the FM85® supplement (Nestlé) (group III). Analyses of carbohydrates, protein, lipids, calcium, phosphorus, sodium, osmolality and caloric content were performed, considering a significant difference p < 0.05. RESULTS: Groups I, II, and III showed, respectively, the following results: protein = 1.81, 2.38 and 1.96 g/dL (p < 0.001); carbohydrates = 6.70, 7.25 and 10.06 g/dL (p = 0.006); fat = 3.75, 3.75 and 3.73 g/dL (p = 0.96); calcium = 36.92, 44.75 and 79.37 mg/dL (p = 0.001); phosphorus = 20.02, 23.28 and 56.30 mg/dL (p = 0.02); sodium = 14.32, 14.40 and 20.33 mEq/L (p = 0.143); osmolality = 391.45, 412.47 and 431.00 mOsmol/kgH2O (p = 0.074); and caloric content = 67.78, 72.27 and 81.65 kcal (p = 0.001). CONCLUSION: The studied additives differ significantly from the commercial additive FM85® in some of its components, and its composition may or may not meet the quantity of nutrients suggested by the most recent recommendations.

consequence of delayed fixation on subsequent preservation of urine cells

Degenerative changes caused by delays in urine preservation contribute to false-negative and false-positive interpretation of urothelial disease in cytology. The aim of this study is to assess whether the delay of fixation of urine samples makes any significant difference to urine cytology and morphology, and the limit of acceptability of delay for routine use in the hospital laboratory. Three cell collection fluids were evaluated by analyzing the preservation and degeneration of cells in urine samples. In this study, 50 voided urine specimens were taken at random from females complaining of vaginal discharge. Each specimen was divided into three sterile containers. The first was immediately centrifugated and the deposit was smeared onto a cleaned micro slide and immediately fixed into 95% ethyl alcohol for 15 minutes. The remaining two were prepared in the same manner, however, the second after two hours of collection and the third after four hours of collection. The degree of degeneration and thus the preservation were assessed by a table of chosen criteria, then ranked and analyzed using Friedman's nonparametric test, at p=0.05. The results showed a significant difference between the preservation and the delay in urine fixation, p<0.0001. Any delay in fixation of urine specimen for cytology affects the preservation of cells, which may result in miss diagnosis. It is recommended that urine samples for cytology should be fixed immediately after collection

Estudio comparativo de dos técnicas de plastinación / Two different plastination techniques comparative study

La técnica de plastinación es utilizada ampliamente para la mejor preservación de las piezas cadavéricas utilizadas en docencia. Nuestra unidad la está utilizando desde el año 2002, pero hemos notado que la calidad de las muestras no es la óptima, produciéndose gran retracción de éstas. Por tal motivo, se diseñó una trabajo que compara la técnica de plastinación de nuestra unidad, con otro proceso, el cual se basa en el protocolo utilizado por la Universidad de Murcia. Utilizamos 24 muestras frescas de riñones y hemiencéfalos de vacuno, riñones y corazones de cerdo y miembro inferior humano, las cuales fueron fijadas con alcohol etílico, formalina y solución fijadora Universidad de los Andes. Fueron aplicadas dos diferentes técnicas de plastinación: una basada en el protocolo utilizado hasta ese momento en nuestro laboratorio de plastinación (técnica A) y otro proceso similar al publicado por la Universidad de Murcia (técnica B). Se evaluaron los porcentajes de variación de peso, ancho, largo, espesor y perímetro de las diferentes piezas plastinadas. También se procedió a fijar las muestras en diferentes soluciones (alcohol, formalina 10 por ciento y solucion fijadora Universidad de los Andes), pesar y medir sus dimensiones. El porcentaje de pérdida de peso promedio fue 63,1 por ciento para la técnica A y 37,9 por ciento para la técnica B (p<0,01); las mediciones de las disminuciones en el largo, ancho y grosor de las muestras también fueron mayores en las piezas sometidas a la técnica A, siendo todos los porcentajes de pérdidas estadísticamente significativos. Al comparar las muestras según los diferentes métodos de fijación con la técnica B no hubo diferencias estadísticamente significativas. La plastinación de muestras basadas en la técnica de Murcia obtiene muestras de mejor calidad y menos retracción. No influiría en el proceso de plastinación el método de fijación.

The plastination technique is used extensively for the improved preservation of cadavers used for teaching. Our unit has been using this technique since 2002, but we have noticed that sample quality is not optimal, given the large retraction of samples. Therefore, a project was designed that compares our department's plastination technique, with another process, based on the protocol used by Murcia University. We used 24 fresh bovine kidneys and brain samples, pigs' kidneys and hearts and human lower limbs; all of which were fixed with ethyl alcohol, formalin and De los Andes University's fixation solution. Two different plastination techniques were applied: one based on the protocol used in our laboratory (technique A) and another one similar to that published by Murcia University (technique B). We evaluated the body weight percentage, width, length, thickness and perimeter of the different plastinated pieces, as well as measuring the same variables in each fixation solution (alcohol, formalin fixative solution 10 percent and Universidad de los Andes). The average weight loss percentage was 63.1 percent for technique A and 37.9 percent for technique B (p <0.01). The decrease measurements in length, width and thickness were also higher under technique A, all percentages being statistically significant. When comparing the samples according to different fixation methods, there were no significant differences. Plastination based on Murcia University's technique obtained better quality samples with less shrinkage. The plastination process was not influenced by the fixation method.

Conservación de cepas vacunales de Leptospira en nitrógeno líquido / Preservation for vaccinal strains of Leptospira at liquid nitrogen

INTRODUCCIÓN: las cepas de Leptospira interrogans que integran la vacuna cubana vax-SPIRAL® son conservadas en medio semisólido de Fletcher, el cual garantiza el mantenimiento de la viabilidad de las células por pocos meses. OBJETIVO: en este estudio, se evaluó la crioconservación en nitrógeno líquido de 6 variantes de crioprotectores como método de conservación a largo plazo de las cepas de Leptospira interrogans, utilizadas como antígenos en la vacuna antileptospirósica vax-SPIRAL®. MÉTODOS: la viabilidad se evaluó periódicamente según el rendimiento celular alcanzado por las cepas recién descongeladas en medio EMJH. La estabilidad de la virulencia fue estimada en hámster. La antigenicidad antes y después de la crioconservación fue comparada mediante microaglutinación, frente a una batería de antisueros de referencia correspondientes a los serogrupos vacunales. RESULTADOS: solo el empleo de dimetilsulfóxido a 2,5 y 5 por ciento, y glicerol 2,5 por ciento como agentes crioprotectores permitió una rápida recuperación de las 3 cepas, tras 19 meses de crioconservación sin afectación de su virulencia y antigenicidad. CONCLUSIONES: los resultados obtenidos demuestran la factibilidad de la crioconservación en nitrógeno líquido con un apropiado agente crioprotector, como método de conservación de cepas vacunales de Leptospira.

INTRODUCTION: Leptospira interrogans strains of Cuban vaccine vax-SPIRAL®are preserved in Fletcher's semi-solid medium, which guarantees the preservation of the cell viability for a few months. OBJECTIVE: to evaluate the cryopreservation at liquid nitrogen of 6 different cryoprotectors as a long-term preservation method for Leptospira interrogans strains used as antigens in vaccine vax-SPIRAL® against leptospirosis. METHODS: viability was systematically evaluated according to the cell yield of the recently thawed strains in EMJH medium. Virulence stability was estimated in hamsters and antigenicity was evaluated by microscopic agglutination test using reference antisera from vaccinal serogroups. RESULTS: the use of 2.5 percent and 5 percent dimethyl sulfoxide, and 2.5 percent glycerol allowed quick recovery of the three strains without virulence or antigenicity depletion after 19 months of cryopreservation. CONCLUSIONS: the results showed the feasibility of the cryopreservation at liquid nitrogen using suitable cryoprotectant as a preservation method for vaccinal strains of Leptospira.

Efeitos do timol sobre os parâmetros urinários envolvidos na formação de cálculos / Effects of thymol on urinary parameters related to lithogenesis

INTRODUÇÃO: Em estudo anterior, demonstramos que a acidificação ou alcalinização de amostras de urina no momento de entrega do material ao laboratório em comparação a amostras coletadas com conservantes não alterou os resultados urinários de parâmetros relacionados à investigação metabólica de litíase renal como o oxalato (OxU), cálcio (CaU), magnésio (MgU), ácido úrico (AcUrU) e creatinina (CreatU), com exceção do citrato (CitU), cujo valor foi discretamente menor. OBJETIVO: Avaliar se a adição de timol, por meio de sua ação antibacteriana, é capaz de prevenir a redução do CitU em amostras acidificadas 24 horas após a coleta, em relação às pré-acidificadas, sem interferir na determinação dos outros parâmetros urinários. MÉTODOS: 40 voluntários sadios coletaram uma amostra isolada de urina que foi dividida em quatro alíquotas de 10 ml contendo timol (1 g/l). Na primeira, o conservante ácido (HCl 6 N, 20 ml/l) foi adicionado imediatamente após a coleta e na segunda, somente após 24 horas. Além do CitU, nessas amostras também foram determinados OxU, CaU e MgU. Na terceira e quarta alíquotas, um conservante alcalino (NaHCO3, 5g/l) foi adicionado imediatamente ou 24 horas após a coleta para determinação do AcUrU. RESULTADOS: Na presença de timol, não se observou variação significante do CitU entre as urinas pré ou pós-acidificadas (577 ± 490 mg/l vs. 575 ± 501 mg/l). Os valores dos demais parâmetros também não sofreram alteração. CONCLUSÃO: A adição prévia de timol às amostras de urina permite que todos os parâmetros urinários litogênicos possam ser determinados numa mesma amostra, reduzindo o custo e o desconforto de múltiplas coletas de urina de 24 horas.

INTRODUCTION: In a previous study, we demonstrated that acidification or alkalinization of urine samples upon delivery of the material to the laboratory in comparison with samples with preservatives did not alter the results of urinary parameters related to the metabolic investigation into renal lithiasis such as oxalate (OxU), calcium (CaU), magnesium (MgU), uric acid (AcUrU) and creatinine (CreatU), with the exception of citrate (CitU), whose value was slightly lower. OBJECTIVE: To evaluate if the addition of thymol, through its antibacterial effect, is able to prevent the reduction of CitU observed in samples acidified 24 hs after collection in comparison with pre-acidified ones without interfering in the determination of other urinary parameters. METHODS: Forty (40) healthy volunteers collected a single spot urine sample, which was divided into four aliquots of 10 ml containing thymol (1 g/l). In the first sample, the acid preservative (HCl6N, 20 ml/l) was added immediately after collection and in the second, only after 24hs. OxU, CaU, CitU and MgU were determined. In the third and fourth aliquots, an alkali preservative (NaHCO3,5 g/l) was added immediately or 24 hs after collection for AcUrU determination. RESULTS: In the presence of thymol, there was no significant variation in CitU values between pre-or post-acidified samples (577±490 mg/l vs. 575±501 mg/l). The values of other parameters also remained unchanged. CONCLUSION: The prior addition of thymol to urine samples allows the determination of all lithogenic urinary parameters in the same sample, reducing the cost and inconvenience of multiple 24-hour urine collections.

Viabilidade, confirmação taxonômica e detecção enzimática de espécies de Acremonium preservadas sob óleo mineral na Coleção de Culturas University Recife Mycology / Viability, taxonomic confirmation and enzymatic detection of Acremonium species preserved under mineral oil in the URM Culture Collection

Enzimas hidrolíticas secretadas por fungos têm um papel importante na patogenicidade das infecções. Objetivando avaliar a atividade enzimática foram testados 31 isolados de Acremonium mantidos na Coleção de Culturas University Recife Mycology. Fragmentos das culturas foram transferidos para caldo glicosado para reativação e posterior crescimento em meio ágar batata dextrose, para verificar viabilidade, pureza e confirmação taxonômica pela observação das características macroscópicas e microscópicas. Para detecção enzimática foram utilizados substratos de caseína do leite e gelatina para protease, amido para amilase e lecitina de soja para fosfolipase. Das 31 culturas, 26 (83,9 por cento) mantiveram-se viáveis e 24 (92,3 por cento) foram confirmadas taxonomicamente. Das 24 culturas, 12 (50 por cento) apresentaram atividade proteásica, duas (16,7 por cento) em caseína do leite, uma (8,3 por cento) em gelatina e nove (75 por cento) em ambos os substratos; 16 (66,7 por cento) degradaram amido. Nenhuma cultura apresentou atividade fosfolipásica. Conclui-se que espécies de Acremonium são capazes de produzir enzimas envolvidas na patogenicidade das infecções fúngicas.

Hydrolytic enzymes secreted by fungi play an important role in the pathogenesis of infection. With the aim of evaluating the enzymatic activity, 31 isolates of Acremonium stored in the University of Recife Mycology (URM) Culture Collection were tested. Culture fragments were transferred to glycoside broth for reactivation and further growth in potato dextrose agar medium in order to investigate viability and purity and to confirm the taxonomy through observing the macroscopic and microscopic characteristics. To detect enzymes, milk casein and gelatin were used as substrates for proteinase, starch for amylase and soy lecithin for phospholipase. Among the 31 cultures, 26 (83.9 percent) remained viable and 24 (92.3 percent) were confirmed taxonomically. Out of these 24 cultures, 12 (50 percent) presented proteinase activity, of which two (16.7 percent) were on milk casein, one (8.3 percent) on gelatin and nine (75 percent) on both substrates; 16 (66.7 percent) degraded starch. None of the cultures presented phospholipase activity. It was concluded that Acremonium species are able to produce enzymes that are involved in the pathogenicity of fungal infections.

Collection, preservation and forwarding of biolgical samples for toxicological analysis in medicolegal autopsy cases : A review.

Collection of proper autopsy specimen is an essential step in the process of toxicology case work¹. Improper collection of these specimens can greatly alter or negate chemical and toxicological analysis. This article is an update about the standard methods of biological specimen collection procedures for toxicological analysis which will be helpful for the forensic pathologist and forensic scientists.

Long-term storage and viability of Ochlerotatus albifasciatus (Diptera: Culicidae)

The viability of Ochlerotatus albifasciatus (Macquart) eggs stored at room temperature and at 5ºC was studied over 31 months. After 12, 18 and 31 months of storage, eggs were acclimatized at 22ºC for ten days, and then inundated twice every seven days. The effect of the storage period on the percentage of hatching was analyzed by one way ANOVA. Differences on the hatching response between the first and second flooding were analyzed by paired t-test. Differences on the hatching response between the two storage conditions were analyzed by Mann-Whitney rank test. Results showed that (1) Oc. albifasciatus eggs were able to survive and hatch over 31 months; (2) the percent hatching of eggs stored at 5ºC was higher than that of eggs stored at room temperature; and (3) low temperatures and long periods without water favor installment hatching.

Evaluation of phenotypic and genotypic alterations induced by long periods of subculturing of Cryptococcus neoformans strains

Cryptococcus neoformans is an encapsulated fungal organism that can cause disease in apparently immunocompetent, as well as immunocompromised, hosts. Since 1930, successive subculture has been used to preserve C. neoformans isolates in our Fungus Collection. In the 1970s, some of these Fungus Collection samples were selected to be subjected to a different methods of maintenance - that of lyophilized. Our objective was to analyze C. neoformans isolates in order to make a comparative evaluation between these two methods of preservation. The overall aim of this study was to qualify the preservation technique used in our mycology laboratory since the technique used might affect the survival, stability and purity of the primary isolates in culture. The samples were analyzed using classical mycology methods and using the randomly amplified polymorphic DNA technique In the analysis of phenotypes and genotypes, the typical characteristics of C. neoformans were found to differ in relation to the different methods of preservation employed. The aim of this study was to demonstrate the importance of selecting the appropriate method of preservation for fungus collections. This selection can affect the survival and purity of the cultures, and preserve the stability of their physiological, biochemical, and genetic characteristics.

Evaluation of preservation methods and simulated multiple infection on the fidelity of real-time PCR detection of Leishmania DNA

To detail the evaluation of a real-time polymerase chain reaction [PCR]-based approach to Leishmania detection. Also to test the fidelity of PCR diagnostics in a series of experiments mimicking infection by two species of Leishmania. Leishmania major [a causal agent of cutaneous leishmaniasis] infected Phlebotomus papatasi sandflies were generated to test the effect of four preservation methods on the fidelity of real-time PCR detection of Leishmania DNA. There was no effect of preservation methods on the sensitivity or specificity of two different assays. The ability of these assays to correctly diagnose cases of multiple infection was tested with artificial double infections created by combining DNA from pairs of Leishmania species [L, major, L. tropica and L, panamensis]. One assay failed to properly diagnose certain double infections but overall the PCR methodologies were robust. These findings provide important reassurances for subsequent real world investigations of Leishmania in vector, reservoir and human populations

Coleta e preservação do líquido pleural e biópsia pleural / Collection and preservation of the pleural fluid and pleural biopsy

As amostras de líquido pleural obtidas por toracocentese para o diagnóstico de transudatos e exsudatos devem obedecer a uma rotina de coleta e preservação para a realização de uma análise laboratorial adequada. Igualmente, fragmentos de biópsia de pleura obtidos para o diagnóstico diferencial dos exsudatos devem ser coletados de forma sistemática com o objetivo de otimizar o diagnóstico e facilitar a instituição da terapêutica adequada.

The samples of pleural fluid obtained by thoracentesis for the diagnosis of transudates and exudates shall follow a routine of collection and preservation for an appropriate laboratorial analysis. Equally, fragments of pleura biopsy obtained for the differential diagnosis of the exudates should be collected in a systematic way in order to optimize the diagnosis and facilitate the institution of appropriate therapeutics actions.

Viability and molecular authentication of Coccidioides immitis strains from culture collection of the Instituto Oswaldo Cruz, Rio de Janeiro, Brazil

Vinte cepas de Coccidioides immitis foram avaliadas.Cinco das 20 cepas preservadas sob óleo mineral mantiveram-se viáveis, todas as 5 subculturas preservadas em água permaneceram viáveis e nenhuma das 13 subculturas mantidas em solo foi viável. Um produto de PCR de 519 pb do gene csa confirmou a identidade das cepas.