Toxigenic potential of Aspergillus flavus tested in different culture conditions

The objective of the present work was to evaluate the capacity of three isolates of Aspergillus flavus to produce aflatoxin under different culture conditions. This experiment was based on a 2³ factorial design, in which the independent variables were temperature (20-40 °C), incubation time (7-21 days), and the pH (2.0-6.0) in two different synthetic media. The optimal conditions were applied to non-aflatoxigenic isolates previously tested in coconut agar. Aflatoxin B1 was extracted directly from the synthetic cultures with chloroform. Thin Layer Chromatography (TLC) and Photographic Photometry were utilized to identify and quantify the compounds. Preliminary results showed that YES agar was an alternative medium for detecting the toxigenic potential of Aspergillus flavus in the following conditions: pH of 5.2, temperature of 25 °C, and incubation time of 11 days producing 206.05 ng.CFU-1 of aflatoxin B1. Of the 30 non-aflatoxigenic isolates, 12 presented a positive result in the optimal media and conditions tested.

Chlamydospore formation by Paracoccidioides brasiliensis mycelial form

To investigate the role of some adverse environmental conditions in chlamy-dospore formation by the mycelial form of P. brasiliensis, we cultured four P. brasiliensis isolates (18, Bt4, 1183, Pb9) at 25°C within solid agar medium either rich or poor in nutrients. Isolates 18 and 1183 were also cultured under anaerobiosis in a nitrogen atmosphere. Isolate 18 produced great number of terminal and intercalary chlamydospore after 7-10 days of culture in a medium poor in nutrients (2% agar with 0.1% dextrose and polypepton). The three other isolates also produced chlamydospores under the same conditions, but in lower numbers. Chlamydospore production by isolate 18 was abolished when the fungus was cultured in two agar media rich in nutrients (brain heart infusion and potato dextrose agar). Anaerobic incubation of isolate 18 under an atmosphere of N2 showed small mycelial outgrowth with numerous chlamydospores. At the electron microscopical level, the chlamydospores showed one or various nuclei and numerous mitochondria, indicating great potential for further development. Accordingly, chlamydospores produced multiple budding after only 24 h incubation at 35°C. The results demonstrate that under adverse environmental conditions P. brasiliensis mycelial form produces chlamydospores within a short period of time.

Blood culture as a parameter of treatment effectiveness in experimental histoplasmosis of the hamster

The aim of this study was to determine the value of blood culture as a parameter of treatment effectiveness in experimental histoplasmosis. A total of thirty five hamsters, weighing approximately 120g, were inoculated intracardiacly with 0.1 ml of a suspension containing 4 x 10(7) cells/ml of the yeast phase of H. capsulatum. Treatments were started one week after the infection and lasted for 3 weeks. The azoles, (itraconazole, saperconazole and fluconazole) were administered once a day by gavage, at a dose of 8 mg/kg; Amphotericin B was given intraperitonealy every other day at a dose of 6mg/kg. Blood samples (1 ml) were obtained by heart punction from the 4th day after infection and were seeded in Sabouraud honey-agar and BHI-agar. The hamsters that survived were killed one week after treatment completion and the following criteria were considered for treatment evaluation: 1) rate of spontaneous death, at the end of the experience; 2) microscopic examination of Giemsa smears from liver and spleen and 3) determination of CFU in spleen cultures. Amphotericin B was the most effective drug, with negative blood cultures at day 20, negative spleen cultures in all cases and all the animals survived until the end of the study. Fluconazole was the less effective drug, blood cultures were positive during the whole experience, spleen cultures showed a similar average of CFU when compared with the control animals and 42.8% of these animals died. Saperconazole and itraconazole showed a similar activity, with survival of all hamsters and negative blood cultures at 23 and 26 days respectively. Blood culture seems to be valuable parameter for treatments' evaluation in experimental histoplasmosis of the hamster.

Sôbre u'a modificação do meio de Monteverde

It is well known that the culture media used in the presumptive diagnosis of suspiciuous colonies from plates inoculated with stools for isolation of enteric organisms do not always correctly indicate the major groups of enterobacteria. In an effort to obtain a medium affording more exact indications, several media (1-9) have been tested. Modifications of some of these media have also been tested with the result that a satisfactory modification of Monteverde's medium was finaly selected. This proved to be most satisfactory, affording, as a result of only one inoculation, a complete series of basic indications. The modification involves changes in the formula, in the method of preparation and in the manner of storage. The formulae are: A. Thymol blue indicator: NaOH 0.1/N .............. 34.4 ml; Thymol blue .............. 1.6 g; Water .................... 65.6 ml. B. Andrade's indicator. C. Urea and sugar solution: Urea ..................... 20 g; Lactose ................... 30 g; Sucrose ................... 30 g; Water .................... 100 ml. The mixture (C.) should be warmed slightly in order to dissolve the ingredients rapidly. Sterilise by filtration (Seitz). Keep stock in refrigeratior. The modification of Monteverde's medium is prepared in two parts. Semi-solid part - Peptone (Difco) 2.0 g; NaCl 0.5 g; Agar 0.5 g; Water 100.0 ml. Boil to dissolve the ingredients. Adjust pH with NaOH to 7.3-7.4. Boil again for precipitation. Filter through cotton. Ad indicators "A" 0.3 ml and "B" 1.0 ml. Sterilise in autoclave 115ºC, 15 minutes in amounts not higher than 200 ml. Just before using, add solution "C" asseptically in amounts of 10 ml to 200 ml of the melted semi-solid medium, maintained at 48-50ºC. Solid part - Peptone (Difco) 1.5 g; Trypticase (BBL) 0.5 g; Agar 2.0 g; Water 100,00 ml. Boil to dissolve the ingredients. Adjust pH with NaOH to 7.3-7.4. Boils again. Filter through cotton. Add indicators "A" 0.3 ml and "B" 1.0 ml; ferrous ammonium sulfate 0.02 g; sodiun thiosulfate 0.02 g. Sterilise in autoclave 115ºC, 15 minutes in amounts not higher than 200 ml. Just before using, add solution "C" asseptically in amounts of 10 ml to 200 ml of the melted solid medium, maintained at 48-50ºC. Final medium - The semi-solid part is dispensed first (tubes about 12 x 120 mm) in 2.5 ml amounts and left to harden at room temperature, in vertical position. The solid part is dispensed over the hardened semi-solid one in amounts from 2.0 ml to 2.5 ml and left to harden in slant position, affording a butt of 12 to 15 mm. The tubes of medium should be subjected to a sterility test in the incubator, overnight. Tubes showing spontaneous gas bubbles (air) should then be discarded. The medium should be stored in the incubator (37ºC), for not more than 2 to 4 days. Storage of the tubes in the ice-box produces the absorption of air which is released as bubbles when the tubes are incubated at 37ºC after inoculation. This fact confirmed the observation of ARCHAMBAULT & McCRADY (10) who worked with liquid media and the aplication of their observation was found to be essential to the proper working conditions of this double-layer medium. Inoculation - The inoculation is made by means of a long straight needle, as is usually done on the triple sugar, but the needel should penetrate only to about half of the height of the semi-solid column. Indol detection - After inoculation, a strip of sterelized filter papaer previously moistened with Ehrlich's reagent, is suspended above the surface of the medium, being held between the cotton plug and the tube. Indications given - In addition to providing a mass of organisms on the slant for serological invetigations, the medium gives the following indications: 1. Acid from lactose and/or sucrose (red, of yellowsh with strains which reduce the indicators). 2. Gas from lactose and/or sucrose (bubbles). 3. H[2]S production, observed on the solid part (black). 4. Motility observed on the semi-solid part (tubidity). 5. Urease production, observed on solid and semi-solid parts (blue). 6. Indol production, observed on the strip of filter paper (red or purplish). Indol production is not observed with indol positive strains which rapidly acidify the surface o the slant, and the use of oxalic acid has proved to give less sensitive reaction (11). Reading of results - In most cases overnight incubation is enough; sometimes the reactions appear within only a few hours of incubation, affording a definitive orientation of the diagnosis. With some cultures it is necessary to observe the medium during 48 hours of incubation. A description showing typical differential reaction follows: Salmonella: Color of the medium unchanged, with blackening of the solid part when H[2]S is positive. The slant tends to alkalinity (greenish of bluish). Gas always absent. Indol negative. Motility positive or negative. Shigella: Color of the medium unchanged at the beginning of incubation period, but acquiring a red color when the strain is late lactose/sucrose positive. Slant tending to alkalinity (greenish or purplish). Indol positive or negative. Motility, gas and H[2]S always negative. Proteus: Color of the medium generally changes entirely to blue or sometimes to green (urease positive delayed), with blackening of solid part when H[2]S is positive. Motility positive of negative. Indol positive. Gas positive or negative. The strains which attack rapidly sucrose may give a yellow-greenish color to the medium. Sometimes the intense blue color of the medium renders difficult the reading of the H[2]S production. Escherichiae and Klebsiellae: Color of the medium red or yellow (acid) with great and rapid production of gas. Motility positive or negative. Indol generally impossible to observe. Paracoli: Those lactose of sucrose positive give the same reaction as Esherichia. Those lactose or sucrose negatives give the same reactions as Salmonellae. Sometimes indol positive and H[2]S negative. Pseudomonas: Color of the medium unchanged. The slant tends to alkalinity. It is impossible to observe motility because there is no growth in the bottom. Alkaligenes: Color of the medium unchanged. The slant tends to alkalinity. The medium does not alter the antigenic properties of the strains and with the mass of organisms on the slant we can make the serologic diagnosis. It is admitted that this medium is somewhat more laborious to prepare than others used for similar purposes. Nevertheless it can give informations generally obtained by two or three other media. Its use represents much saving in time, labor and material, and we suggest it for routine laboratory work in which a quick presumptive preliminary grouping of enteric organisms is needed.

Biological aspects of the DM28C clone of Trypanosoma cruzi after metacylogenesis in chemically defined media

The biological characterization of the Trypanosoma cruzi clone Dm 28c in terms of its growth in LIT medium, cell-cycle, infectivity to mice and interaction with professional and non-professional phagocytic cells shows that it behaves as a bona fide T. cruzi representant. The biological properties of this myotropic clone do not change according to the origin of the trypomastigote forms (i. e., from triatomines, infected mice, cell-culture or from the chemically defined TAUP and TAU3AAG media). In addition Dm 28c metacyclic trypomastigotes from TAU3AAG medium display a high infectivity level to fibroblasts and muscle cells. Experiments on binding of cationized ferritin to trypomastigotes surface show the existence of cap-like structures of ferritin in regions near the kinetoplast. However the nature and role of these anionic sites remain to be determined. The results indicate that metacyclic trypomastigotes from Dm 28c clone obtained under chemically defined conditions reproduce the biological behaviour of T. cruzi, rendering this system very suitable for the study of cell-parasite interactions and for the isolation of trypanosome relevant macromolecules.

Detection of extracellular proteases from microorganisms on agar plates

We present herein an improved assay for detecting the presence of extracellular proteases from microorganisms on agar plates. Using different substrates (gelatin, BSA, hemoglobin) incorporated into the agar and varying the culture medium composition, we were able to detect proteolytic activities from Pseudomonas aeruginosa, Micrococcus luteus and Serratia marcescens as well as the influence that these components displayed in the expression of these enzymes. For all microorganisms tested we found that in agar-BHI or yeast extract medium containing gelatin the sensitivity of proteinase detection was considerably greater than in BSA-agar or hemoglobin-agar. However, when BSA or hemoglobin were added to the culture medium, there was an increase in growth along with a marked reduction in the amount of proteinase production. In the case of M. luteus the incorporation of glycerol in BHI or yeast extract gelatin-agar induced protease liberation. Our results indicate that the technique described here is of value for detecting extracellular proteases directly in the culture medium, by means of a qualitative assay, simple, inexpensive, straight forward method to assess the presence of the proteolytic activity of a given microorganism colony with great freedom in substrate selection.

Leishmania (Viannia) braziliensis growth in vitro culture relies more on folic acid availability than Leihsmania (Leishmania) amazonensis

We compared the in vitro growth of promastigotes from two Leishmania species in TC-100 and Schneider media. Leishmania (Leishmania) amazonensis replication rates were similar in both tissue culture media and reached maximum rates by 48 h. In contrast Leishmania (Viannia) braziliensis growth was significantly greater in TC-100 but maximum rates were achieved by 96 h. Folic acid appears to be the limiting factor and supplementation of Schneider media with this nutrient improved L. (V.) braziliensis replication rates and decreased the time of maximum replication to 48 h.

Evaluation of a modified culture medium for Borrelia burgdorferi sensu lato

The aim of the present study was to assess the possible use of a modified medium, prepared in the laboratory using the constituents of Barbour-Stonner-Kelly (BSK) medium and medium 199 as base, for the culture of Borrelia strains, comparing the growth of individual strains in this medium and in the BSK-H medium, and the protein profile and antigenic characteristics of Borrelia proteins expressed in these media. A qualitative evaluation of growth of Borrelia species was made with acceptable results (morphology and motility), but during a quantitative evaluation using the three main genospecies of Borrelia, the better results were obtained with a B. burgdorferi sensu stricto strain. The modified medium did not enable the growth of a B. afzelii strain. The protein profile and antigenic characteristic of the expressed proteins in the modified medium were studied with satisfactory results. These results suggest the modified medium as an alternative for the cultivation of Borrelia strains, with some limitations, in poorly-resourced laboratories.

Antifungal activity of extracts from Atacama Desert fungi againstParacoccidioides brasiliensis and identification ofAspergillus felis as a promising source of natural bioactive compounds

Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values≤ 125.0 µg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 µg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, β-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 µg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity.

Embryo rescue from interspecific crosses in apple rootstocks

The objetive of this work was to rescue immature embryos of apple rootstocks Malus prunifolia (Marubakaido) and Malus pumila (M9) after 40-60 days of pollination and to put them into MS culture media supplemented with agar (6 g L-1) and casein hydrolysate (500 mg L-1). Embryos originated from interspecific crosses and open pollination showed differences in the in vitro responses, depending on the female parent, the developmental stage of the embryo, and the culture medium composition. Embryos of the M. pumila rootstock, rescued within 40 days after pollination and put in culture medium supplemented with indolacetic acid (IAA), gibberellic acid (GA3), kinetin and maltose, resulted in a normal development of plantlets. However, embryos originating from hand-pollination, cultivated in medium supplemented with 14 µM IAA, 5 µM kinetin and 1.5 µM Ga3 (MS1), mainly those of M. prunifolia x M. pumila, showed a high percentage of rusted embryos (96.2%). Embryos from open pollination of M. prunifolia and M. pumila formed calluses. It was possible to identify the influence of the female parent by the enhanced development of M. pumila shoots derived from open or hand-pollination. The crossing of responsive species and the use of the technique of embryo culture provided a rapid and uniform germination and, consequently, the development of fully normal seedlings.

Cell suspension culture and plant regeneration of a Brazilian plantain, cultivar Terra

The objective of this study was to establish cell suspension culture and plant regeneration via somatic embryogenesis of a Brazilian plantain, cultivar Terra Maranhão, AAB. Immature male flowers were used as explant source for generating highly embryogenic cultures 45 days after inoculation, which were used for establishment of cell suspension culture and multiplication of secondary somatic embryos. Five semisolid culture media were tested for differentiation, maturation, somatic embryos germination and for plant regeneration. An average of 558 plants per one milliliter of 5% SCV (settled cell volume) were regenerated in the MS medium, with 11.4 µM indolacetic acid and 2.2 µM 6-benzylaminopurine. Regenerated plants showed a normal development, and no visible somaclonal variation was observed in vitro. It is possible to regenerate plants from cell suspensions of plantain banana cultivar Terra using MS medium supplemented with 11.4 µM of IAA and 2.2 µM of BAP.

Production technology for entomopathogenic fungus using a biphasic culture system

The objective of this research was to evaluate combinations of liquid media obtained from agro-industrial residues and by-products, with solid media prepared with mixtures of grains and their derivatives, aiming to increase the production of JAB 02 and JAB 45 isolates of Lecanicillium lecanii. Sporulation, conidial viability and process yield were evaluated as well as the production costs using the JAB 45 isolate as a model system were analyzed. The production of JAB 02 was not increased using the biphasic culture. For JAB 45, some combinations provided an increase in yield, especially cheese whey with wheat bran and wheat grain, with lower production costs. Viability was not influenced by the production method, and the combinations showed no differences in the process yield. The biphasic method is suitable for the production of L. lecanii, and proves to be an appropriate technology to use in mass production by biofactories.

Use of semi-seletive media for detection of Sclerotinia sclerotiorum on bean and soybean seeds

This work was aimed at evaluating the possibility of using bromophenol blue as an indicator for detecting the presence of Sclerotinia sclerotiorum in the seeds of dry-beans (Phaseolus vulgaris) and soybean (Glycine max), through incubation of the seeds on an agar medium and "blotter" substrates. The seeds were artificially inoculated with four S. sclerotiorum isolates, plated on the agar medium, named Neon, and on modified Neon agar media all incubated at 14 and 20 ºC for seven days in the dark. Half of the seeds inoculated were surface desinfested prior to plating on the medium. The seeds showing change of colour in the medium, from blue to light yellow, as well as formation of typical mycelium and sclerotia in some cases, were considered to be infected or contaminated by S. sclerotiorum. The two incubation temperatures compared did not show significant (P<0.05) differences in detection level for most of the isolates tested on the different media. According to results obtained in this study, the Neon agar medium with incubation at 14 or 20 ºC has proved to be a reliable and quick method for the detection of S. sclerotiorum mycelium in naturally infected seeds of bean and soybean.

Callus cell culture of Pothomorphe umbellata (L.) under stress condition leads to high content of peroxidase enzyme

Pothomorphe umbellata (L.) known on Brazil as Caapeba has a number of popular medicinal use, and it has been studied in relation to its pharmacological activity. Peroxidase specific activity (units/mg protein) was evaluated in callus cell culture samples of the P.umbellata, grown in two different MS medium (media 1 and media 2), submitted to 16 hours photoperiod or kept in darkness. Cell growth rate curve showed that the best growth indices were observed when media 2 submitted to the photoperiod regime was used, followed by the same media kept in darkness (stress condition). The results obtained also showed that the cell culture grown under stress conditions (darkness) lead to high content of peroxidase enzyme (an increase of 700% was observed). Kinetic constant values of 3.3 mmol.L-1 and 2,8 sec-1 were obtained for kM and v max,, respectively, using guaiacol as enzyme substrate.

Semi-selective culture medium for Exserohilum turcicum isolation from corn seeds

Northern corn leaf blight, caused by Exserohilum turcicum (Et), is a disease of widespread occurrence in regions where corn, sweetcorn and popcorn are grown. This disease has great potential to cause damage and has been studied for years, but the association of its causal agent with seeds remains unconfirmed. Thus, the availability of a sensitive method to detect and quantify the inoculum in seeds, even at low incidence, is essential. The aim of this study was to develop a method to detect and quantify the presence of the fungus infecting and infesting corn and popcorn seeds. Artificially and naturally infected seeds were employed to develop the medium. The semi-selective medium was composed of carbendazim (active ingredient) (60 mg/L), captan (30 mg/L), streptomycin sulfate (500 mg/L) and neomycin sulfate (600 mg/L) aggregated to the medium lactose casein hydrolysate agar medium. By using this, Et was detected in naturally infected corn seeds, showing 0.124% incidence, in four out of ten analyzed samples. In addition, 1.04 conidia were detected per infested seed. By means of isolation, pathogenicity test, morphological characterization and comparison with descriptions of the species in the literature, the fungus isolated from the seeds was confirmed to be Et. Both infection and infestation were considered low; thus, for studies of Et detection in corn seeds, the use of semi-selective medium and more than 1,200 seeds/sample is suggested.