Adhemar Purchio

A new fluorescent viability test for fungi cells

The fluorescein diacetate-ethidium bromide (FDA-EB) fluorescence method, primarily used to determine viability of mammalian cells, was applied to several fungi species. Living fungi cells produced fluorochromasia, i.e., an intracellular accumulation of fluorescein which could be easily visualized as a green color under the U.V. microscope. Dead cells showed a red bright color due to ethidium bromide penetration. The FDA-EB test can be successfuly employed to assay yeast and yeast like cells viability since a good correlation was observed between this assay and the colony count technique. The main advantages of FDA-EB test are its speed, high sensitivity and simplicity.

Evaluation of a fluorescent method (fluorescein diacetate and ethidium bromide solution) in the study of the viability Cryptococcus neoformans strains

The effectiveness of the fluorescent viability test (fluorescein diacetate-FDA and ethidium bromide-EB-solution) compared to the plaque counting test (Miles & Misra M & M) was performed on 10 samples of Cryptococcus neoformans cultivated in Sabouraud dextrose agar at 25 °C. The optimum incubation period of 50 minutes was determined. Growth curves of the fungal strains studied based on the mean cell number were drawn for both the FDA & EB and M & M methods. The statistical evaluation (Students T test) of the average sum of the viable cell counts showed that the FDA-EB method is more sensitive than the M & M test for the studied species. The growth curves of the samples usually followed a homogeneous pattern comparable to other non-dimorphic fungi.

Fungos contaminantes e produtores de aflatoxinas em castanha do Pará (Bertholletia excelsa HUMB. & BONPL 1808)

One hundred samples of Brazil nuts from the States of Amazonas and Sao Paulo, were analysed mycologically and toxicologically for aflatoxins B1 and G1. Three hundred and twelve fungi were isolated of which 91 were of the genus Aspergillus, 83 of the genus Penicillium and the remainder included 23 different genera. Amongst the Aspergillus samples, 26 were producers of aflatoxin, distributed into three species: Aspergillus flavus Link (18); Aspergillus parasiticus Speare (7) and Aspergillus fresenii Subram (1). Of the 100 extracts of analysed nuts, three were afiatoxin-positive.

Aflatoxins B1, M1 and aflatoxicol in tissues and urine of calves receiving aflatoxin.

Liver and kidney tissues and urine from calves chronically or acutely intoxicated by aflatoxin were surveyed to detect the presence of aflatoxins B1, M1 (AFB1, AFM1) and aflatoxicol (AFL). Aflatoxins B1, M1, and aflatoxicol were not found in the liver, kidney or urine from animals intoxicated by chronic forms. However in a calf that received a single dose of 0.8 mg of AFB1/kg of live weight and one submitted to a single dose of 1.8 mg of AFB1/kg of live weight detectable levels of aflatoxins occurred in tissues and urine.

Método fluorescente (diacetato de fluoresceína e brometo de etídeo) para o estudo da viabilidade de Cryptococcus neoformans em líquor

The utilization of the fluorescent method (fluorescein diacetate DF and ethidium bromide BE), to verify the viability of fungal cells, was studied in 40 samples of liquor, from patients with neurocryptococcosis. For removing leukocytes and red blood cells, which produce interfering fluorescence, good results were obtained with 0.3% saponin solution. After processing of liquor, 0.1 ml aliquots of resulting suspension were mixed to equal volumes of fresh DF-BE solution. The best incubation period for staining was 30 minutes, resulting in good differentiation between viable (green fluorescence) and non viable (red fluorescence) cells.

Variations in the levels of aflatoxin in cows milk consumed in the city of São Paulo, Brazil

One hundred samples of commercially available cows milk, collected in the state of São Paulo, from July 1979 to September 1981, were analysed to determine the levels of aflatoxins M1 and M2 by the method of the AOAC. This investigation was also undertaken in 50 samples of cows milk from two farms located in the Médio Vale do Paraiba, from animals which had ingested stored feed. Aflatoxin M1 was detected in only one sample of commercially available cows milk, while those from the farms were found to contain a minimum of 0.1 microgram/l and a maximum of 1.68 microgram/l.

Dimorphism of Scopulariopsis brevicaulis: Morphogenesis of the mould to yeast phase

The dimorphism of Scopulariopsis brevicaulus was induced in malt agar (Oxoid), at 37°C and 5–10% rate of CO2. Six cultures of S. brevicaulus were converted from hyphae to yeast-like cells by the following processes: One was that the hyphae formed intercalar and terminal chlamydoconidia-like cells. These cells increased in size, gained double contour and gave rise to budding cells. The other process was via conidia that assumed yeast characteristics. So, a transformation cycle M → Y was proposed. The yeast-like form presented slow reversion to the mould phase, standing, many times, in transition forms. A high concentration of sugars, peptone, temperature of cultivation of 37°C and 5–10% rate of CO2 seem to be favourable factors for the maintenance of the yeast phase. Ethanol, E.D.T.A., potassium nitrate, temperature of 25°C and aerobiosis are considered favourable factors to conversion of the mould phase.

Ocorrência de aflatoxinas em castanhas do Pará (Bertholletia excelsa, Humb. & Bonpl., 1808)

The occurrence of aflatoxins was investigated in one hundred samples of Brazil nuts (Bertholletia excelsa Humb. & Bonpl.) from producers and from several seed marketing posts, in the region of Manaus, State of Amazonas and Sao Paulo, state of Sao Paulo. The samples were classified according to four types of product presentation: naturally within the shell; dehydrated within the shell; dehydrated without shell and within the chestnut bur. Three samples were found contaminated with aflatoxins types B1 and G1. The toxicity levels for B1 were regarded as: medium (0,1 ppm) and very high (2.25 ppm); those ofaGI were regarded low (0.075) and very high (1.5 ppm).

Search for B-1 aflatoxin and similar fluorescent compounds in ‘Misso’

Following previous studies on the investigation of B-1 Aflatoxin in food samples (4, 5) we report in this paper the results obtained using thin layer chromatography (TLC) with three different solvent systems and spectrophotometric analysis in order to differentiate actual B-1 Aflatoxin and Aflatoxin-like substances. Twenty ‘Misso’ samples were studied and we could detect Aflatoxin in 3 of them. An Aflatoxin-suspected substance was also isolated. No Aflatoxin-producing Aspergillus was isolated from the samples analysed.

Atividade antibacteriana e antifúngica de espécies de Gnetum.

Este trabalho trata da determinacao da atividade antibiotica dos extratos de Gnetum paniculatum e G. schwackeanum e dos constituintes quimicos isolados deste ultimo como resveratrol, gnetina C e E, os quais foram testados contra varios bacterias e fungos. O extrato de Gnetum schwackeanum e todas as substâncias dele isoladas foram ativos a Staphylococcus aureus, S. epidermis e Mycobacterium smegmatis. Resveratrol e gnetina C sao ativos contra Candida albicans, mas somente gnetina C possui atividade em relacao a Candida parapsilosis e Saccharomyces cerevisiae. O derivado sintetico de gnetina E nao mostrou nenhuma atividade. O extrato de G. paniculatum, e completamente inativo a bacteria e fungos o que sugere que a atividade de G. schwackeanum deve-se a presenca dos hiroxiestilbenos e seus derivados, uma vez que G. paniculatum nao contem esses tipos de substâncias.