Concept: Agar plate
The large-scale production of nematophagous fungi as agents of biological control is one of the main challenges to be commercially used. In order to improve growth of microorganism in a culture medium, the addition of growth inducer is common. At the moment, the action of their addition in the mycelia growth and sporulation rate of nematophagous fungi is not known. The purpose of this trial was to evaluate the sporulation rate of Duddingtonia flagrans by adding two growth inducers, meso-inositol and Tween 80, both at 0.5 % in a traditional culture medium Sabouraud glucose agar (SGA) and also in a traditional culture medium enriched with wheat flour and milk powder. From a traditional sterile culture of D. flagrans, four groups were made: SGA; Sabouraud glucose agar-meso-inositol 0.5 %; Sabouraud glucose agar-Tween 80 0.5 %; and Sabouraud glucose agar-enriched (SGA-E). These media were placed at a constant temperature of 27 °C for 4 weeks. Following this, chlamydospores were gently rinsed off with sterile water and counted using a Neubauer haematocytometer to estimate the number of chlamydospores per millilitre of water. The addition of meso-inositol 0.5 % to SGA promoted a significant increase (p < 0.05) in chlamydospore production obtaining an average of 51,715,000 chlamydospores per Petri dish. The highest chlamydospore concentration was observed in the SGA-E in comparison with SGA (p < 0.01) obtaining an average of 208,760,000 chlamydospores. The aim of this study was to obtain basic knowledge regarding the effect of enriched culture medium and growth-inducing meso-inositol and Tween 80 on mycelial growth and production of chlamydospores.
To compare the blood agar (BA), sabouraud dextrose agar (SDA) and chocolate agar (CA) for the isolation of fungi in patients with mycotic keratitis. Corneal Scrapings of 229 patients with clinically diagnosed microbial keratitis were inoculated on BA, SDA, CA. The culture media were evaluated for the rate and time taken for the fungal growth. Seventy six of 229 patients had fungal keratitis. Fungus grew on BA in 60/76(78.9 %), on SDA in 76/76 (100 %), on CA in 40/76(52.6 %) patients. The fungi which grew on BA (60/76) also grown on SDA at the same time. The colony morphologies of different fungi were better on SDA than BA/CA. Among the different culture media, SDA is essential for the isolation fungi in patients with mycotic keratitis.
Tetrazolium salts (TTZ), such as tetrazolium violet (TV), have been widely used for microbiological studies. The formation of the colored formazan product due to bacterial reduction of the uncolored reagent is extensively exploited to stain cells or colonies in agar or on filters. But an important toxic effect of tetrazolium salts on bacteria exists that limits their use at high concentrations, impairing the efficient staining of the colonies. This is especially the case for Salmonella spp. where we observed, using a classic photometric approach and mathematical modeling of the growth, an important impact of tetrazolium violet on the apparent growth rate below the inhibitory concentration. In this study, we demonstrate that adding magnesium to the medium in the presence of TV leads to a significant increase in the apparent growth rate. Moreover, when higher TV concentrations are used which lead to total inhibition of Salmonella strains, magnesium addition to the culture media allows growth and TV reduction. This effect of magnesium may allow the use of higher TTZ concentrations in liquid growth media and enhance bacteria detection capabilities.
Endemic brucellosis threatens wild herds of wood bison (Bison bison athabascae) in and around Wood Buffalo National Park, the largest genetic reserve of wood bison in the world. The overall goal of our project was to produce and preserve disease-free embryos for the purpose of conserving the genetic diversity of this species. The aim of the present experiment was to determine the effectiveness of washing procedures for removing Brucella bacteria from in vivo-derived wood bison embryos exposed in vitro to the pathogen. Wood bison cows were given 300mg im of Folltropin diluted in 0.5% hyaluronan on the day of follicle wave emergence (Day 0) and 100mg im of hyaluronan on Day 2, and then given 2500IU im of hCG on Day 5 and inseminated 12 and 24h later. Embryos were collected on Day 13. The experiment was done in 6 replicates (n=4 bison/replicate) and an average of 9 embryos/replicate were collected. Zona pellucida-intact embryos were kept in holding medium (PBS+2% fetal calf serum) and transported to a Biosafety Level 3 laboratory at the International Vaccine Centre, University of Saskatchewan. Embryos were transferred through 5 aliquots of holding medium to remove any contaminant before exposure to Brucella. Embryos were divided equally into 2 Petri dishes (representing later wash groups with v. without antibiotics) containing 2.7mL of holding medium (n=2 to 7 embryos per dish/replicate). In a Class II biosafety cabinet, Brucella abortus biovar 1 (1×10(7) to 1×10(9)CFUmL(-1) in 0.3mL) was added to each Petri dish and incubated for 2h at 37°C in 8% CO2. A sample of holding medium was taken before exposure and after incubation for culture as negative and positive controls, respectively. After incubation, embryos in each Petri dish were subjected to a 10-step washing procedure (according to the IETS Manual, 2010) using wash medium (PBS+0.4% BSA) without antibiotics or with antibiotics (100IUmL(-1) of penicillin+100μgmL(-1) of streptomycin). The embryo wash medium was cultured at wash steps 1, 3, 6, and 9. After the tenth wash, the zona pellucida of each embryo was ruptured mechanically using a glass pipette and embryos were cultured individually. Culturing of samples was done on sheep blood agar and specific identification of Brucella organisms was done by PCR. Brucella abortus was detected in 3 embryos from the group washed in medium without antibiotics (3/27), whereas all embryos washed in medium with antibiotics were culture negative (0/27). Brucella abortus was not detected in wash media after the third wash in either group (with or without antibiotics). In summary, Brucella abortus was removed from 89% of in vitro-exposed wood bison embryos using the washing procedure without antibiotics, and from 100% using the washing procedure with antibiotics. Results validate the embryo washing technique for producing Brucella-free wood bison embryos.
Inducing an axenic culture of the edible cyanobacterium Arthrospira (Spirulina) platensis using differential filtration alone is never successful; thus, it has been thought that, in non-axenic cultures, a portion of contaminating bacteria is strongly associated with Arthrospira cells. However, examination of the behavior of these bacteria during filtration revealed that they were not associated with Arthrospira cells but with aggregates of exopolysaccharides present in the medium away from the Arthrospira cells. Based on this finding, a rapid and reliable method for preparing axenic trichomes of A. platensis was established. After verifying the axenicity of the resulting trichomes on enriched agar plates, they were individually transferred to fresh sterile medium using a handmade tool, a microtrowel, to produce axenic cultures. With this technique, axenic cultures of various A. platensis strains were successfully produced. The technique described in this study is potentially applicable to a wider range of filamentous cyanobacteria.
ABSTRACT We investigated the application capabilities of a laser optical sensor, BARDOT (bacterial rapid detection using optical scatter technology) to generate differentiating scatter patterns for the 20 most frequently reported serovars of Salmonella enterica. Initially, the study tested the classification ability of BARDOT by using six Salmonella serovars grown on brain heart infusion, brilliant green, xylose lysine deoxycholate, and xylose lysine tergitol 4 (XLT4) agar plates. Highly accurate discrimination (95.9%) was obtained by using scatter signatures collected from colonies grown on XLT4. Further verification used a total of 36 serovars (the top 20 plus 16) comprising 123 strains with classification precision levels of 88 to 100%. The similarities between the optical phenotypes of strains analyzed by BARDOT were in general agreement with the genotypes analyzed by pulsed-field gel electrophoresis (PFGE). BARDOT was evaluated for the real-time detection and identification of Salmonella colonies grown from inoculated (1.2 × 10(2) CFU/30 g) peanut butter, chicken breast, and spinach or from naturally contaminated meat. After a sequential enrichment in buffered peptone water and modified Rappaport Vassiliadis broth for 4 h each, followed by growth on XLT4 (~16 h), BARDOT detected S. Typhimurium with 84% accuracy in 24 h, returning results comparable to those of the USDA Food Safety and Inspection Service method, which requires ~72 h. BARDOT also detected Salmonella (90 to 100% accuracy) in the presence of background microbiota from naturally contaminated meat, verified by 16S rRNA sequencing and PFGE. Prolonged residence (28 days) of Salmonella in peanut butter did not affect the bacterial ability to form colonies with consistent optical phenotypes. This study shows BARDOT’s potential for nondestructive and high-throughput detection of Salmonella in food samples. IMPORTANCE High-throughput screening of food products for pathogens would have a significant impact on the reduction of food-borne hazards. A laser optical sensor was developed to screen pathogen colonies on an agar plate instantly without damaging the colonies; this method aids in early pathogen detection by the classical microbiological culture-based method. Here we demonstrate that this sensor was able to detect the 36 Salmonella serovars tested, including the top 20 serovars, and to identify isolates of the top 8 Salmonella serovars. Furthermore, it can detect Salmonella in food samples in the presence of background microbiota in 24 h, whereas the standard USDA Food Safety and Inspection Service method requires about 72 h.
BACKGROUND: Bartonella henselae is a zoonotic, alpha Proteobacterium, historically associated with cat scratch disease (CSD), but more recently associated with persistent bacteremia, fever of unknown origin, arthritic and neurological disorders, and bacillary angiomatosis, and peliosis hepatis in immunocompromised patients. A family from the Netherlands contacted our laboratory requesting to be included in a research study (NCSU-IRB#1960), designed to characterize Bartonella spp. bacteremia in people with extensive arthropod or animal exposure. All four family members had been exposed to tick bites in Zeeland, southwestern Netherlands. The mother and son were exhibiting symptoms including fatigue, headaches, memory loss, disorientation, peripheral neuropathic pain, striae (son only), and loss of coordination, whereas the father and daughter were healthy. METHODS: Each family member was tested for serological evidence of Bartonella exposure using B. vinsonii subsp. berkhoffii genotypes I-III, B. henselae and B. koehlerae indirect fluorescent antibody assays and for bacteremia using the BAPGM enrichment blood culture platform. RESULTS: The mother was seroreactive to multiple Bartonella spp. antigens and bacteremia was confirmed by PCR amplification of B. henselae DNA from blood, and from a BAPGM blood agar plate subculture isolate. The son was not seroreactive to any Bartonella sp. antigen, but B. henselae DNA was amplified from several blood and serum samples, from BAPGM enrichment blood culture, and from a cutaneous striae biopsy. The father and daughter were seronegative to all Bartonella spp. antigens, and negative for Bartonella DNA amplification. CONCLUSIONS: Historically, persistent B. henselae bacteremia was not thought to occur in immunocompetent humans. To our knowledge, this study provides preliminary evidence supporting the possibility of persistent B. henselae bacteremia in immunocompetent persons from Europe. Cat or flea contact was considered an unlikely source of transmission and the mother, a physician, reported that clinical symptoms developed following tick exposure. To our knowledge, this is the first time that a B. henselae organism has been visualized in and amplified from a striae lesion. As the tick bites occurred three years prior to documentation of B. henselae bacteremia, the mode of transmission could not be determined.
While advancements have been made in some areas of pathology with diagnostic materials being screened using image analysis technologies, the reporting of cultures from agar plates remains a manual process. We compared results for 2163 urine cultures read by a reference panel of microbiologists, the routine laboratory process and by an automated plate reading system, APAS® (LBT Innovations, Ltd, South Australia). APAS® detected colonies with a sensitivity of 99.1% and specificity of 99.3% on blood agar while on MacConkey agar, the colony detection sensitivity was 99.4 % with a specificity of 99.3%. The device’s ability to enumerate growth had an accuracy of 89.2% and the morphologic identification of colonies showed a high level of performance for the colony types typical of E. coli and other enteric bacilli. On blood agar, lactose-fermenting colonies were morphologically identified with a sensitivity of 98.9% while on MacConkey agar they were identified with a sensitivity of 99.2%. In this first clinical evaluation, APAS® demonstrated a high performance in the detection, enumeration and colonial classification of isolates when compared with conventional plate reading methods. The device found all cases reported by the laboratory and detected the most commonly encountered organisms found in urinary tract infections.
This paper describes the trends in prevalence of ESBL producing Enterobacteriaceae (ESBL-E) and ESBL genes, measured in five consecutive yearly Point Prevalence Surveys (PPS). All patients present in the hospital and in a day-care clinic (including patients on dialysis) on the day of the survey, were screened for perianal ESBL-E carriage. Perianal swabs were taken and cultured using an enrichment broth and a selective agar plate. Both phenotypic and genotypic methods were used to detect the production of ESBL, presence of ESBL-genes and clonal relatedness. Out of 2,695 patients, 135 (5.0%) were tested ESBL-E positive. The overall ESBL-E prevalence was stable over the years. Overall 5.2% of all ESBL-E were acquired by nosocomial transmission. A relative decrease of CTX-M-1-1-like ESBL genes (from 44 to 25%, p = 0.026) was observed, possibly related to the strong (>60%) decrease in antibiotic use in livestock in our country during the same period.
Microbiologists have been using agar growth medium for over 120 years. It revolutionized microbiology in the 1890’s when microbiologists were seeking effective methods to isolate microorganisms, which led to the successful cultivation of microorganisms as single clones. But there has been a disparity between total cell counts and cultivable cell counts on plates, often referred to as the “great plate count anomaly”, that has long been a phenomenon that still remains unsolved. Here we report that a common practice microbiologists have been employing to prepare agar medium has a hidden pitfall: when phosphate was autoclaved together with agar to prepare solid growth media (PT medium), total colony counts were remarkably lower when compared with those grown on agar plates in which phosphate and agar were separately autoclaved and mixed right before solidification (PS medium). We used a pure culture Gemmatimonas aurantiaca T-27(T) and three representative sources of environmental samples, soil, sediment and water, as inocula and compared colony counts between PT and PS agar plates. There were higher numbers of colony forming units (CFUs) on PS medium compared to PT medium using G. aurantiaca or any of the environmental samples. Chemical analysis of PT agar plates suggested that hydrogen peroxide was contributing to growth inhibition. Comparison of 454 pyrosequences of the environmental samples to the isolates revealed that taxa grown on PS medium were more reflective of the original community structure than those grown on PT medium. Moreover, more hitherto-uncultivated microbes grew on PS than on PT medium.