Solubilization of CaHPO4 and AlPO4 by Aspergillus niger in culture media with different carbon and nitrogen sources

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Culture of mature trophoblastic giant cells from bovine placentomes

The mostly binucleate trophoblast giant cells (TGC) found in bovine placentomes, in addition to synthesizing and releasing hormones play an important role in fetal development and maternal adaptation to pregnancy. Placentomes from early gestation were collected, and for isolation of mature TGC, three cellular disaggregation methods, mechanical (MECH), enzymatic by trypsin (TRYP) or collagenase (COLL) were compared to each other. Further on, the cell survival in culture medium (DMEM) supplemented with either 10% fetal calf serum (FCS) or 10% serum replacement (SR) on culture plates free of any substrate was evaluated over a period of 90 days by trypan blue exclusion. The cells were further characterized by HOECHST 33342 nuclear staining, and immunocytochemical staining with monoclonal antibodies against vimentim and cytokeratin. A mean total rate of TGC survival of 82.56% was recorded. Statistical analysis showed significantly higher survival rates after enzymatic disaggregation with COLL (86.23%) than following MECH (80.38%) or TRYP (80.91%) treatment. Supplementation of DMEM with FCS resulted in significantly higher cellular survival rates (87.13%) when compared to the addition of SR (77.73%). Analysis of the influence of both, disaggregation method and medium supplementation on TGC survival revealed statistically significant differences between the following groups: MECH-SR (71.09%) was significantly lower than all other groups; TRYP-SR (78.03%) was significantly different from all other groups; TRYP-FCS (83.43%) and COLL-SR (84.08%) were significantly lower than MECH-FCS (89.98%) which together with COLL-FCS (88.25%) showed the highest cellular survival rate. In summary, our results show that TGC isolated from early gestation placentomes may be viable for more than 90 days of culture. However, whether these TGC produce placental lactogen throughout this period has yet to be determined. (c) 2006 Elsevier B.V. All rights reserved.

Effect of insulin-like growth factor-1 during in vitro oocyte maturation and in vitro culture of bovine embryos

Avaliaram-se o efeito do IGF-I na maturação in vitro (MIV) (experimento I) e no desenvolvimento embrionário (DE) (experimento II) de oócitos bovinos fecundados in vitro, quanto às taxas de clivagem (TC), de blastocistos (TB) e de eclosão (TE). Para MIV, complexos cumulus-oócitos imaturos foram cultivados em meio TCM-199 suplementado com HEPES, bicarbonato e piruvato de sódio, aditivos, soro fetal bovino (meio B-199) e gonadotrofinas 14U/ml de PMSG e 7U/ml de hCG). Para o desenvolvimento embrionário, os oócitos/zigotos foram cultivados em meio B-199 com células epiteliais do oviduto bovino em suspensão sob óleo de silicone. As condições de cultivo in vitro para ambos os experimentos seguiram os tratamentos: 1- meio B-199 + 200 ng/ml IGF-I; 2- B-199 + 100 ng/ml IGF-I; 3- B-199 + 50 ng/ml IGF-I; 4- B-199 + 10 ng/ml IGF-I; 5- B-199 + 0 ng/ml IGF-I. Todas as culturas foram realizadas a 38,5° C em atmosfera com 5% de CO2 e os dados foram analisados pelo teste do qui-quadrado. No experimento I, não houve diferença (P>0,05) entre os tratamentos quanto às TC, TB e TE, quando o meio de MIV foi suplementado com IGF-I. No experimento II, a adição de IGF-I ao meio de DE resultou em aumento na TC (P<0,05) mas não influenciou a TB e a TE. A adição de 200 ng/ml de IGF-I ao meio DE melhorou a TC (71,1%) quando comparada com a TC dos grupos de 100 ng/ml de IGF-I (57,6%) ou controle (56,7%), entretanto não houve diferença quando comparada com a dos grupos de 50 ng/ml (69,4%) ou 10 ng/ml (73,1%) de IGF-I. Não houve efeito benéfico na adição de 10 a 200 ng/ml de IGF-I nos meios de MIV e de DE com relação ao desenvolvimento de embriões produzidos a partir de oócitos maturados e fecundados in vitro.

Growth and enzymatic responses of phytopathogenic fungi to glucose in culture media and soil

The effect of inoculation of Aspergillus flavus, Fusarium verticillioides, and Penicillium sp. in Dystrophic Red Latosol (DRL) and Eutroferric Red Latosol (ERL) soils with or without glucose on the total carbohydrate content and the dehydrogenase and amylase activities was studied. The fungal growth and spore production in culture medium with and without glucose were also evaluated. A completely randomized design with factorial arrangement was used. The addition of glucose in the culture medium increased the growth rate of A. flavus and Penicillium sp. but not of F. verticillioides. The number of spores increased 1.2 for F. verticillioides and 8.2 times for A. flavus in the medium with glucose, but was reduced 3.5 times for Penicillium sp. The total carbohydrates contents reduced significantly according to first and second degree equations. The consumption of total carbohydrates by A. flavus and Penicillium sp. was higher than the control or soil inoculated with F. verticillioides. The addition of glucose to soils benefited the use of carbohydrates, probably due to the stimulation of fungal growth. Dehydrogenase activity increased between 1.5 to 1.8 times (p <0.05) in soils with glucose and inoculated with the fungi (except F. verticillioides), in relation to soil without glucose. Amylase activity increased 1.3 to 1.5 times due to the addition of glucose in the soil. Increased amylase activity was observed in the DRL soil with glucose and inoculated with A. flavus and Penicillium sp. when compared to control.

Meios de cultura no desenvolvimento de ápices caulinares de mamoneira (Ricinus communis L.) in vitro

A pesquisa da composição do meio de cultura mais adequado à espécie vegetal e ao tipo de explante empregado é o fator de maior relevância da cultura de tecidos. O cultivo de ápice caulinar com recuperação da planta matriz é uma técnica de grande impacto para a propagação de plantas in vitro, regeneração de plantas livres de vírus, conservação de germoplasma e modificação genética. Objetivou-se, neste trabalho, avaliar composições do meio de cultivo para organogênese direta in vitro a partir de ápices caulinares pertencentes à população FCA-UNESP-PB de mamoneira (Ricinus communis L.), com vistas à propagação clonal de genótipos elite. Foram testadas quatro formulações: MS básico (T1), MS modificado 1 (T2), MS modificado 2 (T3) e WPM (T4), em delineamento experimental inteiramente casualizado, com 20 repetições em cada tratamento, sendo a repetição 1 ápice caulinar/frasco. O T3 apresentou-se superior e diferiu significativamente dos outros tratamentos apresentando 35% dos ápices caulinares diferenciados e desenvolvidos; seguiu-se o T2 com 10% e os tratamentos T1 e T4 não apresentaram diferenciação de tecidos. Os resultados permitiram concluir que os balanceamentos de sais minerais nos meios de cultura avaliados, especialmente a relação NO3 / NH4 e ausência de FeSO4.7H2O, indicaram grande influência no desenvolvimento de ápices caulinares de mamoneira.

Viability of human fibroblasts in coconut water as a storage medium

P>AimTo evaluate the effectiveness of a new storage medium for avulsed teeth, coconut water, in maintaining the viability of human fibroblasts.MethodologyCell viability after different time periods was evaluated in the following storage media: coconut water, coconut water with sodium bicarbonate, milk, saline and still mineral water. Human fibroblasts were seeded in Eagle's minimal essential medium (EMEM) supplemented with 7.5% foetal calf serum. After trypsinisation, 100 mu L of culture medium containing approximately 10(4) cells mL(-1) were collected and pipetted into the wells of 96-well plates, which were incubated overnight in 5% CO(2) and 95% air mixture at 37 degrees C. EMEM was then replaced by the storage media and the plates were incubated at 37 degrees C for 1, 2 and 4 h. Cell viability was determined using the neutral red assay. The proportions of viable cells after exposure to the storage media were analysed statistically by anova and the least significant difference (LSD) test (alpha = 5%).ResultsMilk had the greatest capacity to maintain cell viability (P < 0.05), followed by coconut water with sodium bicarbonate and saline. Coconut water was significantly worse at maintaining cell viability compared to milk, coconut water with sodium bicarbonate and saline. The smallest number of viable cells was observed for mineral water (P < 0.05).ConclusionCoconut water was worse than milk in maintaining human fibroblast cell viability.

Effect of culture conditions on the biomass determination by ergosterol of Lentinus crinitus and Psilocybe castanella

Estimating fungal growth is important in processes of soil bioremediation. It has been demonstrated that ergosterol is a good indicator of fungal biomass in solid substrata. In the present study were evaluated the effects upon the ergosterol rate of Lentinus crinitus Berk. and Psilocybe castanella Peck through the culture conditions of these fungi, which are evaluated for the bioremediation of soils contaminated by organochlorates. A good correlation between fungal biomass and ergosterol was observed for both species. The culture conditions did not influence the ergosterol rate of L. crinitus. Yet the ergosterol rate of P. castanella was influenced from 35 days of culture and when grown in the presence of 15.00 g hexachlorobenzene l(-1) of culture medium. So it is possible to estimate growth of both species using ergosterol as indicator in processes of soil bioremediation since the influences observed in the ergosterol rate of P. castanella are considered.

Influence of culture media in viability test of conidia of entomopathogenic fungi

O presente trabalho objetivou investigar se meios de cultura utilizados em teste de viabilidade afetam a germinação de conídios de cinco isolados de Lecanicillium lecanii, cinco de Beauveria bassiana e quatro de Paecilomyces fumosoroseus. Os testes foram realizados em lâminas de microscopia contendo um dos seguintes meios de cultura: Ágar-água (AA), Meio Mínimo (MM), Batata, dextrose e ágar (BDA), Batata, dextrose, ágar e 1% de extrato de levedura (BDAL), Sabouraud, dextrose, ágar e extrato de levedura (SDAL) e Meio Completo (MC). Delimitaram-se três áreas por lâmina e em cada uma aplicou-se 0,05mL de uma suspensão com concentração de 5,5 x 105 conídios ml-1. Para cada isolado foi realizado um bioensaio, com seis tratamentos e cinco repetições. Avaliou-se a germinação 15 horas após incubação, a 26±0,5ºC. Os meios de cultura influíram na capacidade de germinação das três espécies estudadas, ocorrendo variações inter e intraespecíficas. Verificou-se que os meios Completo e BDA proporcionaram as maiores porcentagens de germinação dos isolados de L. lecanii, sendo que e as menores foram obtidas nos meios SDAL e AA. Os meios ricos em nutrientes (BDA, BDAL e Completo) favoreceram a germinação dos isolados de B. bassiana, o que não ocorreu com os meios pobres (AA e MM). Nos meios Completo e BDA foram obtidas as maiores porcentagens de germinação dos isolados de P. fumosoroseus. As menores percentagens, por sua vez, foram obtidas no meio SDAL. Entretanto, alguns isolados apresentaram alta germinação em meios pobres em nutrientes (AA e MM).

AGAR MEDIUM FOR HUMAN UREAPLASMA UREALYTICUM

The authors wish to report a simple medium for the isolation and cultivation of Ureaplasma urealyticum starting from clinical materials. This medium induced growth in eleven of the twelve different serotypes of Ureaplasma urealyticum studied.

Simple method for culture of peripheral blood lymphocytes of Testudinidae.

We developed and optimized a simple, efficient and inexpensive method for in vitro culture of peripheral blood lymphocytes from the Brazilian tortoise Chelonoidis carbonaria (Testudinidae), testing various parameters, including culture medium, mitogen concentration, mitotic index, culture volume, incubation time, and mitotic arrest. Peripheral blood samples were obtained from the costal vein of four couples. The conditions that gave a good mitotic index were lymphocytes cultured at 37°C in minimum essential medium (7.5 mL), with phytohemagglutinin as a mitogen (0.375 mL), plus streptomycin/penicillin (0.1 mL), and an incubation period of 72 h. Mitotic arrest was induced by 2-h exposure to colchicine (0.1 mL), 70 h after establishing the culture. After mitotic arrest, the cells were hypotonized with 0.075 M KCl for 2 h and fixed with methanol/acetic acid (3:1). The non-banded mitotic chromosomes were visualized by Giemsa staining. The diploid chromosome number of C. carbonaria was found to be 52 in females and males, and sex chromosomes were not observed. We were able to culture peripheral blood lymphocytes of a Brazilian tortoise in vitro, for the preparation of mitotic chromosomes.

Effects of ascorbic acid on in vitro culture of bovine preantral follicles

The objective of this study was to evaluate the effects of adding ascorbic acid to the media for in vitro culture of cattle ovarian fragments and to determine their effects on growth activation and viability of early-stage follicles. The ovarian cortex was divided into small fragments; one fragment was immediately fixed (control) and the other fragments were cultured in minimum essential medium (MEM) supplemented or not with various doses of ascorbic acid. Ovarian tissue was processed for histology, transmission electron microscopy (TEM) and immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA). Compared with control fragments, the percentage of primordial follicles was reduced (p < 0.05) and the percentage of growing follicles had increased (p < 0.05) in cultured cortical fragments, independent of the tested medium or incubation time. Furthermore, compared with control tissue, culture of ovarian cortex for 8 days reduced the percentages of healthy, viable follicles (p < 0.05), but not when cultures were supplemented with 25, 50 or 100 μg/ml of ascorbic acid. Ultrastructural and immunohistochemical analysis of 8 day cultured ovarian cortical fragments, however, showed the integrity and viability of follicles only when fragments were cultured in presence of 50 μg/ml of ascorbic acid. In conclusion, this study demonstrated that addition of ascorbic acid to MEM at a concentration of 50 μg/ml not only stimulates the activation of 8 day in vitro cultured cattle primordial follicles and subsequent growth of activated follicles, but also safeguards the viability of these early-stage follicles. © 2012 Copyright Cambridge University Press.

Propagação de Hancornia speciosa Gomes – Apocynaceae, por alporquia e micropropagação

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Propagação vegetativa de Tamarindus indica L

Pós-graduação em Agronomia - FEIS

Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media

The objective of this study was to evaluate the culture of equine bone marrow mononuclear fraction and adipose tissue - derived stromal vascular fraction cells in two different cell culture media. Five adult horses were submitted to bone marrow aspiration from the sternum, and then from the adipose tissue of the gluteal region near the base of the tail. Mononuclear fraction and stromal vascular fraction were isolated from the samples and cultivated in DMEM medium supplemented with 10% fetal bovine serum or in AIM-V medium. The cultures were observed once a week with an inverted microscope, to perform a qualitative analysis of the morphology of the cells as well as the general appearance of the cell culture. Colony-forming units (CFU) were counted on days 5, 15 and 25 of cell culture. During the first week of culture, differences were observed between the samples from the same source maintained in different culture media. The number of colonies was significantly higher in samples of bone marrow in relation to samples of adipose tissue.

Mycelial growth of two Lentinula edodes strains in culture media prepared with sawdust extracts from seven eucalyptus species and three eucalyptus clones

The in vitro mycelial growth of Lentinula edodes strains LE-95/01 and LE-96/18 were evaluated in solid culture media prepared with sawdust extracts from seven eucalyptus species (E. saligna, E. grandis, E. urophylla, E. pellita, E. paniculata, E. citriodora, and E. camaldulensis) and three eucalyptus clones (E. grandis × E. urophylla hybrids). Evaluations were made every 48 hours by means of colony diameter measurements (mean of four transversely-oriented measurements), during ten days of incubation in the dark at 25ºC ±1°C. The experimental design consisted of randomized blocks, and treatment means were compared by Tukey test. The culture medium prepared from E. citriodora sawdust extract was the most promising to grow L. edodes strains LE-96/18 and LE-95/01. L. edodes strain LE-96/18 presented the fastest mycelial growth after incubation for ten days, regardless of sawdust extract type used in the culture medium.