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.

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.

Use of a selective medium with potassium tellurite to follow intestinal colonization of hospitalized patients by drug-resistant Enterobacteriaceae

Nosocomial infections are a relevant factor in complicating the recovery of patients interned for even minor causes. In attempt to determine their origin it is crucial to consider that their origin is of an endogenous nature. Looking for an acessible expression of intestinal colonization we analyzed fecal samples from 3 separate groups of hospital patients collected after different lenghts of time. For practical reasons one group was studied prospectively and two other groups (patients hospitalized for up to 7 days and patients hospitalized for more than 7 days) were compared to one another. We looked for the emergence of tellurite resistance among Enterobacteriaceae using a selective medium. MacConkey potassium tellurite (MCPT). The frequence of prospectively studied patients with tellurite resistant strains was significantly greater after 7 days of hospitalization. For the two other groups, patients with more than 7 days of hospitalization showed a significant increase of bacterial species and of strains with new antimicrobial resistance markers. High molecular weigth plasmids were detected in some of these strains. These data show that the MCPT medium is a useful tool for the investigation of bowel colonization in hospitalized patients by drug-resistant Enterobacteriaceae.

Semi-selective medium for Fusarium graminearum detection in seed samples

Fungi of the genus Fusarium cause a variety of difficult to control diseases in different crops, including winter cereals and maize. Among the species of this genus Fusarium graminearum deserves attention. The aim of this work was to develop a semi-selective medium to study this fungus. In several experiments, substrates for fungal growth were tested, including fungicides and antibiotics such as iprodiona, nystatin and triadimenol, and the antibacterial agents streptomycin and neomycin sulfate. Five seed samples of wheat, barley, oat, black beans and soybeans for F. graminearum detection by using the media Nash and Snyder agar (NSA), Segalin & Reis agar (SRA) and one-quarter dextrose agar (1/4PDA; potato 50g; dextrose 5g and agar 20g), either unsupplemented or supplemented with various concentrations of the antimicrobial agents cited above. The selected components and concentrations (g.L-1) of the proposed medium, Segalin & Reis agar (SRA-FG), were: iprodiona 0.05; nystatin 0,025; triadimenol 0.015; neomycin sulfate 0.05; and streptomycin sulfate, 0.3 added of ¼ potato sucrose agar. In the isolation from seeds of cited plant species, the sensitivity of this medium was similar to that of NSA but with de advantage of maintaining the colony morphological aspects similar to those observed in potato-dextrose-agar medium.

A Semi-Selective agar medium to detect the presence of Xanthomonas axonopodis pv. malvacearum in naturally infected cotton seed

A semi-selective agar medium was developed for detection of Xanthomonas axonopodis pv. malvacearum (Xam) in cotton (Gossypium hirsutum) seed. The basic medium was peptone-sucrose-agar (PSA). Criteria for the semi-selective medium were the typical colony characters of Xam and its pathogenicity on cotton. Several systemic fungicides and antibiotics in different concentrations were tested alone or in combination with others. The final composition of the semi-selective agar medium was established after several attempts in order to inhibit most of the fungal and bacterial saprophytes and favour the development of Xam. It contained PSA + cyclohexamide, cephalexin, pencycuron, triadimenol and tolylfluanid. The bacteria were recovered from naturally infected seeds by the direct plating of 2,000 surface disinfected seeds on the semi-selective medium. The recovery of the pathogen from naturally infected leaf tissues and in dilution plating, on semi-selective medium and on nutrient agar, were comparable. Among the three detection methods tested, the semi-selective medium was found to be the most reliable and quantifiable. Degree of severity of angular leaf spot in the field was not always correlated with the level of infection in the seed. This is the first report of a semi-selective agar medium to detect the presence of Xam in naturally infected cotton seed.

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.

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%).

DETECTION OF VIRULENCE GENES IN ENVIRONMENTAL STRAINS OF Vibrio cholerae FROM ESTUARIES IN NORTHEASTERN BRAZIL

The objectives of this study were to detect the presence of Vibrio cholerae in tropical estuaries (Northeastern Brazil) and to search for virulence factors in the environmental isolates. Water and sediment samples were inoculated onto a vibrio-selective medium (TCBS), and colonies with morphological resemblance to V. cholerae were isolated. The cultures were identified phenotypically using a dichotomous key based on biochemical characteristics. The total DNA extracted was amplified by PCR to detect ompW and by multiplex PCR to detect the virulence genes ctx, tcp, zot and rfbO1. The results of the phenotypic and genotypic identification were compared. Nine strains of V. cholerae were identified phenotypically, five of which were confirmed by detection of the species-specific gene ompW. The dichotomous key was efficient at differentiating environmental strains of V. cholerae. Strains of V. cholerae were found in all four estuaries, but none possessed virulence genes.

Bacterial spot and early blight biocontrol by epiphytic bacteria in tomato plants

The objective of this work was to evaluate in vitro and in vivo biocontrol of bacterial spot (Xanthomonas vesicatoria) and early blight (Alternaria solani) by the epiphytic bacteria Paenibacillus macerans and Bacillus pumilus. Tomato plants were previously sprayed with epiphytic bacteria, benzalkonium chloride and PBS buffer and, after four days, they were inoculated with A. solani and X. vesicatoria. To determine the phytopathogenic bacteria population, leaflet samples were collected from each treatment every 24 hours, for seven days, and plated on semi-selective medium. The effect of epiphytic bacteria over phytopathogens was performed by the antibiosis test and antagonistic activity measured by inhibition zone diameter. The epiphytic and benzalkonium chloride drastically reduced the severity of early blight and bacterial spot in comparison to the control (PBS). In detached leaflets, the epiphytic bacteria reduced in 70% the number of phytopathogenic bacteria cells in the phylloplane. The antibiosis test showed that the epiphytic bacteria efficiently inhibit the phytopathogens growth. In all the bioassays, the epiphytic bacteria protect tomato plants against the phytopathogens

Detection of drechslera avenae in oat seeds

The fungus Drechslera avenae, the causal agent of Helminthosporium leaf spot on oats (Avena sativa), survives as mycelium in crop residues and in infected seeds. In trials carried out in the laboratory, ten methods were evaluated for their efficiency to detect D. avenae in oat seeds. In each experiment, groups of two or three methods were compared to a standard protocol, in which seeds were placed in Petri dishes containing the Reis selective medium and incubated at 25±2 °C for ten days. Data were submitted to analysis of variation and the means of the methods were compared using the Dunnett test at the 5% significance level. Overall, the highest levels of seed infection by D. avenae were observed on oat seeds plated in the osmotic, the oat-agar and the Reis media, or on seeds subjected to heat treatment previous to incubation in malt-agar. Therefore, these methods should be recommended for detection of D. avenae in oat seed testing.

Incidence of pectolytic erwinias associated with blackleg of potato in Rio Grande do Sul

Erwinia carotovora subsp. atroseptica (Eca), E. carotovora subsp. carotovora (Ecc) and E. chrysanthemi (Ech) may cause potato (Solanum tuberosum) blackleg. To determine the occurrence of these pathogens in the conditions found in the State of Rio Grande do Sul (RS), potato plants showing blackleg symptoms were harvested from 22 fields in nine counties in Serra do Nordeste, Planalto, Depressão Central, and Grandes Lagoas, from September to December of 1999 (Spring-Summer season). Green pepper (Capsicum annuum) fruits were used as a host to enrich for pectolytic erwinia from potato stems with blackleg symptoms. Bacteria were subsequently isolated on non-selective medium. Isolates that were Gram-negative, facultatively anaerobic, and pitted crystal-violet-pectate medium were tested for biochemical traits to identify the species and subspecies. Four hundred strains were identified as either Eca, Ecc or Ech. Although the three erwinias were found in RS potato fields, only three strains of Ech were found in one field. Frequencies of Eca and Ecc were 55 and 42%, respectively. Eight strains could not be assigned based on the biochemical characterization.

Meio de cultura semi-seletivo para detecção de Curtobacterium flaccumfaciens pv. flaccumfaciens em solo e sementes de feijoeiro

A murcha-de-curtobacterium, causada pela bactéria Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), é um importante problema para o cultivo de feijão (Phaseolus vulgaris L.) na região Centro-Sul do Brasil. A principal forma de disseminação da bactéria é por sementes contaminadas. Embora a ocorrência desta doença seja relativamente recente no Brasil, Cff tem sido disseminado rapidamente nas regiões produtoras de feijão do país. Este trabalho teve como objetivo ajustar o meio de cultura CNS (Corynebacterium Nebraskense Selective Medium) para recuperação e detecção de Cff em solo e sementes de feijoeiro. Suspensões provenientes da lavagem de amostras de solo e sementes contaminadas pela bactéria foram plaqueadas no meio de cultura CNS - modificado. Os ajustes realizados no meio de cultura possibilitaram a recuperação eficiente de Cff presente em solo e sementes contaminadas. As modificações estabelecidas para o meio CNS foram a redução da concentração de sulfato de polimixina para 16 mg/l, exclusão do componente ciclohexamida e substituição do fungicida Daconil 2787-F (530 mg/ml de chlorothalonil) pelo Dacostar 500 (500 mg/ml de chlorothalonil).

Influence of the medium (strongly acid or basic) over the electrochemical properties of the nitrate selective electrode

A nitrate selective electrode was prepared for use in an aggresive medium (high acidic or basic concentration). It is demonstrated that the depending E graph with respect to pNO3- has not a Nernstian response in concentration acidic range upper 0.1 mol/L H2SO4. The observed behaviour is supposed to be due to the formation of a dimeric anion HN2O6-.

Selective and non-extractive spectrophotometric determination of cefdinir in formulations based on donor-acceptor complex formation

Cefdinir has broad spectrum of activity and high prescription rates, hence its counterfeiting seems imminent. We have proposed a simple, fast, selective and non-extractive spectrophotometric method for the content assay of cefdinir in formulations. The method is based on complexation of cefdinir and Fe under reducing condition in a buffered medium (pH 11) to form a magenta colored donor-acceptor complex (λ max = 550 nm; apparent molar absorptivity = 3720 L mol-1 cm-1). No other cephalosporins, penicillins and common excipients interfere under the test conditions. The Beer's law is followed in the concentration range 8-160 µg mL-1.

Occurrence of Campylobacter jejuni in dog faeces from the streets of a Southern Chilean city

Dog faeces collected from the streets of a southern Chilean city were cultured on selective media for thermophilic Campylobacters. Campylobacter jejuni (biotype 1) was isolated from 53 (35.3%) of 150 samples. The use of an enrichment medium enhanced in 20.8% the isolation rate of this bacteria.