Growth curves of Leishmania braziliensis braziliensis Promastigotes and surface antigen expression before and after adaptation to Schneider's drosophila medium as assessed by anti-Leishmania human sera

Leishmania braziliensis braziliensis(MHOM/BR/75/M2903) was grown in Schneider's Drosophila medium. In one set of experiments promastigotes were already adapted to the medium by means of serial passages whereas in the second cells were grown in a biphasic medium and transfered to the liquid. Growth was more abundant for culture medium adapted cells; degenerate cells in small numbers as well as dead ones were present from day 5 for promastigotes adapted to liquid medium and from day 3 for newly adapted cells. Synthesis of surface antigens differed according to length of cell culture as assessed by the titer of five mucocutaneous leishmaniasis sera on subsequent days. Five days of culture for cells already adapted to the culture medium and 3 days for newly adapted ones were judged to be the best for the preparation of immunofluorescence antigens.

Induction of iron regulated proteins during normal growth of Neisseria meningitidis in a chemically defined medium

The expression of iron regulated proteins (IRPs) in vitro has been obtained in the past by adding iron chelators to the culture after bacterial growth, in the presence of an organic iron source. We have investigated aspects concerning full expression of the meningococcal IRPs during normal growth, in defined conditions using Catlin medium, Mueller Hinton and Tryptic Soy Broth (TSB). The expression of IRPs varied between different strains with respect to Ethylenediamine Di-ortho-Hidroxy-phenyl-acetic acid (EDDA) concentrations, and according to culture medium, and also between different lots of TSB. For each strain, a specific set of IRPs were expressed and higher EDDA concentrations, or addition of glucose, or use of different culture media did not resulted in a differential expression of IRPs. We were not able to grow N. meningitidis under normal growth conditions using Desferal. We looked for a good yield of outer membrane vesicles (OMVs) expressing IRPs in iron-deficient Catlin medium containing EDDA and Hemin. Culture for 32 h at 30ºC after growing for 16 h at 37ºC supported good bacterial growth. Bacterial lysis was noted after additional 24 h at 30ºC. Approximately 4 times more OMVs was recoverable from a culture supernatant after 24 h at 30ºC than from the cells after 16 h at 37ºC. The IRP were as well expressed in OMVs from culture supernatant obtained after 24 h at 30ºC as from the cells after 16 h at 37ºC.

Use of selective medium for Burkholderia cepacia isolation in respiratory samples from cystic fibrosis patients

Burkholderia cepacia colonizes cystic fibrosis (CF) patients. We evaluated the impact of the use of a selective medium in the rate of B. cepacia recovery from respiratory samples of CF patients. During a 6-month period, respiratory samples were collected from 106 CF patients and cultivated on selective media including a B. cepacia selective medium. Confirmation of the identity of B. cepacia isolates was carried out by species specific PCR and determination of genomovar status performed by a sequential PCR approach. Results of B. cepacia isolation during this period were compared to the preceding two years, when the sample processing was identical except for the lack of the B. cepacia selective medium. B. cepacia was isolated in 11/257 (4.2%) of the samples using the selective medium, in contrast with the preceding two years, when it was isolated in 6/1029 samples (0.58%), p < 0.0001. Identity of all 11 isolates was confirmed by PCR and genomovar determination was accomplished in all but one isolate. These results suggest that the use of a selective medium increases recovery rate of B. cepacia from respiratory samples.

Reproduction of Leishmania (Leishmania ) infantum chagasi in conditioned cell culture growth medium

Leishmanias can be produced by inoculation in conditioned McCoy cell culture growth medium (CGM). Leishmania (Leishmania) infantum chagasi (100 parasites) grown in NNN medium was inoculated in 2.5 mL CGM, kept in plates (24 wells) and its multiplication was observed for five days (120 hours). After day 5, the medium was saturated with the flagellate forms of the parasite (promastigotes). The reproduction of the leishmanias was observed every 24 hours and the number of parasites was calculated by counting the parasites in a drop of 10 µ L and photomicrographied. So the number of Leishmanias was adjusted to 1 mL volume. The advantage of the technique by isolation of Leishmania in CGM demonstrated in this study is its low cost and high efficacy even with a small quantity of parasites (10² promastigotes) used as inoculum. Additionally, isolation of the leishmania can be obtained together with an increase in their density (180 times) as observed by growth kinetics, within a shorter time. These results justify the use of this low-cost technique for the isolation and investigation of the behavior and multiplication of Leishmania both in vertebrates and invertebrates, besides offering means of obtaining antigens, whether whole antigens (leishmanias) or the soluble antigens produced by the parasites which may be useful for the production of new diagnostic kits.

CO-INFECTION OF DENGUE VIRUS BY SEROTYPES 1 AND 4 IN PATIENT FROM MEDIUM SIZED CITY FROM BRAZIL

SUMMARY The natural co-infection with dengue virus can occur in highly endemic areas where different serotypes have been observed for many years. We report one case of DENV-1/DENV-4 co-infection in human serum detected by molecular tests. Phylogenetic analysis of the sequences obtained indicated the presence of genotype V and II for DENV-1 and DENV-4, respectively.

The comparison of the Butzler medium, filtration technique and their association with isolation of Campylobacter ssp

Campylobacter was isolated in 178 out of 622 stool samples (200 porcine, 220 bovine, and 202 canine). From these 178 samples, the microorganism was identified in 64 samples (36%) isolated only in Butzler selective medium (BSM), 34 samples (19%) using filtration technique (FT), and in 80 samples (45%) using both BSM and FT. Comparison of the proportion of positivity using both techniques showed a significant value (c2 = 9,184; p > 0.001); BSM (36%) being more efficient than FT (19%). The use of both techniques yielded the highest isolation positivity (45%).

Medium-term cryopreservation of rabies virus samples

Introduction The cryopreservation of rabies virus has been described in detail in the literature. To date, little information is available on the use of cryoprotective agents for cold preservation of this virus, and the available data focus only on short-term virus preservation. In this study, we investigated the medium-term cryopreservation of samples of rabies virus using different cryopreservation protocols. Methods The cryopreservation protocols for the rabies virus samples were performed at -20°C and were divided according to the variables of time and cryoprotectant type used. The laboratory tests (intracerebral inoculation of mice, viral titration and direct immunofluorescence) were performed at regular intervals (360 and 720 days) to assess the viability of the viral samples according to the different preservation techniques used. Results After 1 year of cryopreservation, the fluorescence intensity of intracellular corpuscles of the rabies virus and the median survival time of the mice differed between the positive controls and the treatments with the cryoprotectants. After 2 years, most of the samples subjected to the cryopreservation protocols (including the controls) did not produce fluorescence. However, the virus samples exposed to the cryoprotectant sucrose (68% solution) responded positively in the direct immunofluorescence assay and in the intracerebral inoculation of the mice. Conclusions Medium-term cryopreservation of the rabies virus inactivates the viral sample. However, the cryoprotectant agent sucrose (68%) produces a preservative effect in cryopreserved rabies virus samples.

Abundance of Conepatus chinga (Carnivora, Mephitidae) and other medium-sized mammals in grasslands of southern Brazil

Between January 2007 and December 2010, the abundance of medium-sized mammals was studied, with special focus on the Molina's hog-nosed skunk, Conepatus chinga (Molina, 1782), at four locations in southern Brazil. In this study, transect line methodology was used to obtain data for Distance Analyses. Transects were traveled by car at night, searching with spotlights along the edges of secondary roads in agricultural landscapes. Along 1,811 km, we obtained 620 observations of 20 mammal species. The most common species was the exotic European hare, Lepus europaeus (Pallas, 1778); the highest abundance estimated for South America was observed in one of the study areas, where its density was estimated as 32 individuals/km². Carnivores were the most commonly recorded mammals, represented by 10 species and comprising 51% of all observations. Molina's hog-nosed skunk occurred in all study areas, but occurred in sufficient numbers to obtain density estimates in only two of the areas. We estimated 1.4 to 3.8 individuals/km², in the first density estimate made by the transect method for a member of Conepatus in the Neotropics. These values are similar to those estimated for North American species of Mephitidae. In Brazil, C. chinga is apparently more abundant in the Pampa biome than in the grasslands of the Atlantic Forest. For two other carnivores, Lycalopex gymnocercus (Fisher, 1814) and Cerdocyon thous (Linnaeus, 1766), we estimated preliminary densities that were similar to those previously cited for different regions.

Diversity and habitat preference of medium and large-sized mammals in an urban forest fragment of southwestern Amazon

We assessed the species composition and abundance of medium and large-sized mammals in an urban forest fragment in the Brazilian Amazon, and recorded the preference of some species for particular phytophysiognomies. We placed nine transects with 20 sand plots each in three phytophysiognomies: open rainforest with a dominance of bamboos (OFB), open rainforest with palm trees (OFP), and dense rainforest (DF). We calculated species abundance as the number of records/plot.day, in a total of 2,700 plots.day. We recorded twelve mammal species; Sylvilagus brasiliensis (Linnaeus, 1758) and Dasyprocta fuliginosa (Wagler, 1831) were the most abundant. The results differed among phytophysiognomies: DF presented the highest mammal diversity, whereas the species composition of OFP was less similar than that of other phytophysiognomies. Rodents showed higher preference for OFP and Sylvilagus brasiliensis was more abundant in OFB. The study area showed high species richness, with the occurrence of mesopredators, but there was a predominance of common species adaptable to disturbed environments, which reflects the severe isolation degree of the forest fragment and the hunting pressure that is still present.

A selective medium for the isolation of the acinetobacter genus bacteria

A selective and differencial medium was developed for the isolation of Acinetobacter genus bacteria. This Acinobacter Agar Medium (p.H + 7.4) contains in grams per litre: thiotone, 10; yeast extract, 3; naC1, 5; saccharose, 10; mannitol, 10; sodium citrate, 0.5; sodium desoxycholate, 0.1; crystal violet, 0.00025; phenol red, 0.04 and agar-agar 15. This medium has the advantage of inhibiting the growth of cocci and Gram-positive bacilli, by the use of sodium citrate and sodium desoxycholate associated with the crystal violet; and of differentiating the Gram-negative bacilli from the Enterobacteriaceae, through the fermentative activity upon the saccharose and/or mannitol, contrasting with the complete inactivity of the Acinetobacter genus bacteria over those substances.

Liquid nitrogen cryopreservation of Bracoccidioides brasiliensis in Fava's Netto medium

The aplicability of Fava's Netto medium in the liquid nitrogen cryopreservation technique of Paracoccidioides brasiliensis cells was demonstrated.

The change of behavior of two strains of Leishmania after cultivation in a defined medium

Attempts have been made to characterize two strains of Leishmania that became infective to golden hamsters only after they had been maintained for several years in a chemically defined culture medium. Observations were made on the growth rates of promastigotes in vitro, course of infection in hamsters, morphology of amastigotes, and electrophoretic mobility patterns of eight isoenzymes. Information was obtained about the buoyant densities of n-DNA and k-DNA, and one strain was tested against monoclonal antibodies. The identity of both strains remains obscure.

Medium-term protocols for in vivo evaluation of chemical modifiers of carcinogenesis

Cancer development is a long-term multistep process which allows interventional measure before the clincial disease emerges. the detection of natural substances which can block the process of carcinogenesis is a important as the identification of anti-tumoral drugs since they might be used in chemoprevention of cancer in high-risk groups. In vivo rodent models of chemical caecinogenesis have been used to study plant-derived inhibitors of carcinofenesis such as indols, coumarins, isothiocyanates, flavones, phenols and allyl-sulfides. Since the standard in vivo rodent bioassay is prolonged and expensive, shorter reliable protocols are needed. Two in vivo medium-term protocols for evaluation of modifiers of carcinogenesis are presented, one related to liver and the other to bladder cancer. Both protocols use rats, last 8 and 36 weeks and are based on the two-step concept of carcinogenesis: initiation and promotion. The protocols use respectively the development of altered foci of hepatocytes expressing immunochistochemically the placental form of gluthation S-transferase and the appearence of pre-neoplastic urothelium and papillomas as the "end-points". the use of these protocols for detection of plantpderived inhibitors of carcinogenesis appear warranted.