Providing inoculum for Didymella bryoniae studies: the effect of light spectrum and storing at low temperature

Abstract The in vitro sporulation of Didymella bryoniae is of great importance for studies that require pure inoculum and in large quantities. Thus, the objectives of this study were to identify the best condition for D. bryoniae sporulation combining different light spectra (UV-A or UV-B light, white light, and continuous dark), with distinct culture media (PDA, V8, ML, and PDAB) and, to evaluate fungus survivability stored at -20°C over time. The fungus samples were only able to sporulate when subjected to the UV-B light treatment, regardless of the culture medium. The highest appearance of spores conidium type was observed in the PDAB medium, and the lowest production occurred in the ML medium. Reproductive structures, such as perithecia and pycnidia, were observed in all culture media. However, there was considerable variation in the amount of each structure between the different culture media. The ML and V8 media showed a greater number of perithecia and the PDA and PDAB media presented a greater proportion of pycnidia compared to perithecia. The storage duration at -20°C did not affect mycelial growth or mycelial growth rate. In conclusion, the UV-B light is essential for D. bryoniae in vitro sporulation. Moreover, the culture medium composition influences the type of fungal structure produced, as well as spores size and quantity. Freezing at -20°C is an efficient technique that can be used to store D. bryoniae for at least five months without loss of viability.

Resumo A esporulação de Didymella bryoniae in vitro é de grande importância para estudos que requerem inóculo puro e em grandes quantidades. Assim, os objetivos deste estudo foram identificar a melhor condição para esporulação de D. bryoniae combinando diferentes espectros de luz (luz UV-A ou UV-B, luz branca e escuro contínuo) com distintos meios de cultura (PDA, V8, ML e PDAB) e, avaliar a sobrevivência do fungo armazenado a -20°C ao longo do tempo. As amostras de fungo só esporularam quando submetidas ao tratamento com luz UV-B, independentemente do meio de cultura. Maior aparecimento de esporos do tipo conídio foi observado no meio PDAB, e a menor produção ocorreu no meio ML. Estruturas reprodutivas, como peritécios e picnídeos, foram observadas em todos os meios de cultura. No entanto, houve uma variação considerável na quantidade de cada estrutura entre os diferentes meios de cultura. Os meios ML e V8 apresentaram maior número de peritécios e os meios PDA e PDAB apresentaram maior proporção de picnídeos em relação aos peritécios. A duração do armazenamento a -20°C não afetou o crescimento micelial ou a taxa de crescimento micelial. Em conclusão, a luz UV-B é essencial para a esporulação de D. bryoniae in vitro. Além disso, a composição do meio de cultura influencia o tipo de estrutura fúngica produzida, bem como o tamanho e a quantidade dos esporos. O congelamento a -20°C é uma técnica eficiente que pode ser usada para armazenar D. bryoniae por pelo menos cinco meses sem perda de viabilidade

Providing inoculum for Didymella bryoniae studies: the effect of light spectrum and storing at low temperature / Fornecendo inóculo para estudos de Didymella bryoniae: o efeito do espectro de luz e armazenamento em baixa temperatura

Abstract The in vitro sporulation of Didymella bryoniae is of great importance for studies that require pure inoculum and in large quantities. Thus, the objectives of this study were to identify the best condition for D. bryoniae sporulation combining different light spectra (UV-A or UV-B light, white light, and continuous dark), with distinct culture media (PDA, V8, ML, and PDAB) and, to evaluate fungus' survivability stored at -20°C over time. The fungus samples were only able to sporulate when subjected to the UV-B light treatment, regardless of the culture medium. The highest appearance of spores conidium type was observed in the PDAB medium, and the lowest production occurred in the ML medium. Reproductive structures, such as perithecia and pycnidia, were observed in all culture media. However, there was considerable variation in the amount of each structure between the different culture media. The ML and V8 media showed a greater number of perithecia and the PDA and PDAB media presented a greater proportion of pycnidia compared to perithecia. The storage duration at -20°C did not affect mycelial growth or mycelial growth rate. In conclusion, the UV-B light is essential for D. bryoniae in vitro sporulation. Moreover, the culture medium composition influences the type of fungal structure produced, as well as spores' size and quantity. Freezing at -20°C is an efficient technique that can be used to store D. bryoniae for at least five months without loss of viability.

Resumo A esporulação de Didymella bryoniae in vitro é de grande importância para estudos que requerem inóculo puro e em grandes quantidades. Assim, os objetivos deste estudo foram identificar a melhor condição para esporulação de D. bryoniae combinando diferentes espectros de luz (luz UV-A ou UV-B, luz branca e escuro contínuo) com distintos meios de cultura (PDA, V8, ML e PDAB) e, avaliar a sobrevivência do fungo armazenado a -20°C ao longo do tempo. As amostras de fungo só esporularam quando submetidas ao tratamento com luz UV-B, independentemente do meio de cultura. Maior aparecimento de esporos do tipo conídio foi observado no meio PDAB, e a menor produção ocorreu no meio ML. Estruturas reprodutivas, como peritécios e picnídeos, foram observadas em todos os meios de cultura. No entanto, houve uma variação considerável na quantidade de cada estrutura entre os diferentes meios de cultura. Os meios ML e V8 apresentaram maior número de peritécios e os meios PDA e PDAB apresentaram maior proporção de picnídeos em relação aos peritécios. A duração do armazenamento a -20°C não afetou o crescimento micelial ou a taxa de crescimento micelial. Em conclusão, a luz UV-B é essencial para a esporulação de D. bryoniae in vitro. Além disso, a composição do meio de cultura influencia o tipo de estrutura fúngica produzida, bem como o tamanho e a quantidade dos esporos. O congelamento a -20°C é uma técnica eficiente que pode ser usada para armazenar D. bryoniae por pelo menos cinco meses sem perda de viabilidade

Providing inoculum for Didymella bryoniae studies: the effect of light spectrum and storing at low temperature.

The in vitro sporulation of Didymella bryoniae is of great importance for studies that require pure inoculum and in large quantities. Thus, the objectives of this study were to identify the best condition for D. bryoniae sporulation combining different light spectra (UV-A or UV-B light, white light, and continuous dark), with distinct culture media (PDA, V8, ML, and PDAB) and, to evaluate fungus' survivability stored at -20°C over time. The fungus samples were only able to sporulate when subjected to the UV-B light treatment, regardless of the culture medium. The highest appearance of spores conidium type was observed in the PDAB medium, and the lowest production occurred in the ML medium. Reproductive structures, such as perithecia and pycnidia, were observed in all culture media. However, there was considerable variation in the amount of each structure between the different culture media. The ML and V8 media showed a greater number of perithecia and the PDA and PDAB media presented a greater proportion of pycnidia compared to perithecia. The storage duration at -20°C did not affect mycelial growth or mycelial growth rate. In conclusion, the UV-B light is essential for D. bryoniae in vitro sporulation. Moreover, the culture medium composition influences the type of fungal structure produced, as well as spores' size and quantity. Freezing at -20°C is an efficient technique that can be used to store D. bryoniae for at least five months without loss of viability.

Quantitation of T2 lesion load in multiple sclerosis with magnetic resonance imaging: a pilot study of a probabilistic neural network approach.

RATIONALE AND OBJECTIVES: To quantitate multiple sclerosis (MS) lesions in the brain by using computerized techniques. METHODS: MS lesions from five patients were quantitated with magnetic resonance (MR) imaging by using three approaches: a probabilistic neural network (PNN) approach, a semiautomated method that uses a bifeature space approach with operator intervention at each section, and the "gold standard" of manual outlining of lesions. Each patient underwent two MR studies in 1 day. RESULTS: The PNN approach allows reasonable quantitation of large data sets with minimal operator input. The mean intraobserver error for the PNN approach was competitive with the more time-consuming bifeature space approach (5.2% vs 4.4%, respectively). On average, both computer assisted methods performed better than the manual method (mean intraobserver error, 10.1%). CONCLUSION: The agreement between the two computerized quantitation approaches was good. The number of interactive steps was substantially reduced with the PNN technique, leading to minimal operator intervention time.

Automated determination of left ventricular volume curves from bi-plane digital angiography without explicit use of edge detection algorithms.

Automated computation of left ventricular (LV) global and regional function using contrast angiography has not yet become a routine procedure with the advent of digital cardiac imaging systems. We describe a new technique to compute LV volume curves which does not require the use of manual or semi-automated detection of endocardial borders and provides on-line implementation of volumetric curves and computation of pressure volume loops during catheterization. The approach uses the concepts of variable entropy (or information) of left ventricular images throughout the cardiac cycle. LV volume curves are computed with an interpolation scheme using those LV volume curves of a patient data base which are associated with the closest variation in entropy in the RAO projection to the analyzed patient data according to a simple metric. Computed LV volume curves were correlated with those obtained with manual tracing. Left ventricular ejection fraction (LVEF), time to end systole (TES) and angiographic cardiac output (CO) were compared to those obtained with the manual method. Results using a data base of 365 patients revealed excellent correlation (r = 0.97) between manually derived volume curves and volume curves computed with the automated technique within a large range of LVEFs. In 87% of all cases the computed LVEF values were found within +/- 10% of the value obtained with the gold standard method. The systolic phase of the volume curves showed that 81% of all cases had the same accuracy. The TES showed much more variation due to undersampling of the cardiac cycle in time (r = 0.71).

Computation of left ventricular volume curves from gated blood pool studies without explicit use of edge detection algorithms: concise communication.

A new technique has been developed to compute left ventricular (LV) time activity curves from gated blood pool (GBP) studies without the use of manual, semiautomated or fully automated edge detection algorithms. The method utilizes the correlation of entropy calculated from the counts of a fixed region of interest covering the left ventricle during a cardiac cycle to compute the LV volume curve for a new patient. The new LV volume curve is obtained through interpolation of those volume curves of a data base which are associated with the closest variations in normalized entropy to the new one. The computed LV time activity curves agree with those obtained from manual or fully automated outlines of the left ventricle within 9 percent for the selected set of 67 patients demonstrating the potential of the method. The accuracy of calculated LV volume curves can be improved theoretically to any degree by increasing the number of cases in the data base of known statistical feature vectors associated with the LV images and LV volume curves. The new method for computation of LV curves is very efficient and robust when compared to traditional techniques.

Automated discrimination and quantification of idiopathic pulmonary fibrosis from normal lung parenchyma using generalized fractal dimensions in high-resolution computed tomography images.

RATIONALE AND OBJECTIVES: We computed generalized fractal dimensions for high-resolution computed tomography (HRCT) images to investigate their value in the discrimination and quantification of idiopathic pulmonary fibrosis (IPF) from normal lung parenchyma. METHODS: A probability distribution that was based on the pixel value in each image was used to compute capacity, information, and higher fractal dimensions for a series of 52 HRCT slices obtained from four patients. Qualitative classification of normal, mild, moderate, and severe IPF cases was achieved by computing the following parameter: DD = D0 - 2D1 + D2, where D0, D1, and D2 represents the capacity, information, and pair correlation dimensions, respectively. A multiple linear regression analysis using morphometric quantification for the set of 52 slices was tested for all possible combinations of the parameters D0, D1, D2, and D3. The generalizability of the model was tested by predicting the extent of IPF for each patient from a regression model computed with the remaining slices in the database. RESULTS: The best regression results were obtained using the independent parameters D1 and D2 to quantify the extent of diseased lung parenchyma. The technique was tested with 48 slices from 12 new patients. The results indicated that the extent of IPF could be predicted within the confidence limits given by the regression analysis. CONCLUSION: The extent of IPF can be predicted well within the 90% confidence interval given by the model. The width of the confidence interval decreases as the number of slices used in the linear regression model increases. This operator-independent quantitative technique may be useful in the follow-up of patients with IPF.

Quantitation of grey matter, white matter, and cerebrospinal fluid from spin-echo magnetic resonance images using an artificial neural network technique.

An operator independent technique has been developed to quantitate the volume of white matter (WM), grey matter (GM), and cerebrospinal fluid (CSF) using spin-echo magnetic resonance images. Using skull stripped spin-echo images, CSF was removed using an automated thresholding technique. The bimodal histogram of the remaining images was used to train a perceptron and a single hidden layer neural network to output the percentage of GM and WM in the image. The output values were compared with those of a semiautomated technique employing a least square fitting technique [graduated nonconvexity algorithm (GNC)] applied to the bimodal histogram. This semiautomated technique allowed for intervention by the radiologist. Fourteen normal volunteers with eight contiguous slices each were analyzed. The individual percentages of WM, GM, and CSF of 40 slices from five subjects not used in the training set as well as the total percentages of GM, WM, and CSF in each individual were predicted using the two artificial network architectures. GM, WM, and CSF percentages were predicted within 7% for individual slices while total percentages of WM, GM, and CSF were computed accurately with an absolute error of less than 5% when compared to the semiautomated technique involving a trained neuroradiologist.