Automated detection and quantification of Enchytraeus crypticus (Oligochaeta: Enchytraeidae) in tropical artificial soil using image analysis.

The effects of toxic substance in soil matrices are evaluated by assessing adult worm survival and reproduction. Throughout the test, hundreds of juvenile potworms can be found. The current method for Enchytraeus crypticus quantification in soil samples is a laborious and time-consuming procedure that involves manual counting. The present work proposes a method for quick and reliable counting of E. crypticus by using an automated image analysis algorithm applied to soil images. Comparisons between automated and manual methods conducted in double-blind trials involving a large, routine batch of tropical artificial soil samples revealed no statistically significant differences for a wide range of worm densities. The proposed method overcomes time-consuming counts in manual methods and is suited to be deployed routinely for soil toxicity studies involving large batches of samples.

Label-free detection and enumeration of Giardia cysts in agitated suspensions using in situ microscopy.

Laboratory procedures performed in water treatment studies frequently require the characterization of (oo)cyst suspensions. Standard methods commonly used are laborious, expensive and time-consuming, besides requiring well-trained personnel to prepare samples with fluorescent staining and perform analysis under fluorescence microscopy. In this study, an easy cost-effective in situ microscope was assessed to acquire images of Giardia cysts directly from agitated suspensions without using any chemical labels or sample preparation steps. An image analysis algorithm analyzes the acquired images, and automatically enumerates and provides morphological information of cysts within 10 min. The proposed system was evaluated at different cyst concentrations, achieving a limit of detection of ~30 cysts/mL. The proposed system overcomes cost, time and labor demands by standard methods and has the potential to be an alternative technique for the characterization of Giardia cyst suspensions in resource-limited facilities, since it is independent of experts and free of consumables.

Morphometric quantification of a pseudohyphae forming Saccharomyces cerevisiae strain using in situ microscopy and image analysis.

Yeast morphology and counting are highly important in fermentation as they are often associated with productivity and can be influenced by process conditions. At present, time-consuming and offline methods are utilized for routine analysis of yeast morphology and cell counting using a haemocytometer. In this study, we demonstrate the application of an in situ microscope to obtain a fast stream of pseudohyphae images from agitated sample suspensions of a Saccharomyces cerevisiae strain, whose morphology in cell clusters is frequently found in the bioethanol fermentation industry. The large statistics of microscopic images allow for online determination of the principal morphological characteristics of the pseudohyphae, including the number of constituent cells, cell-size, number of branches, and length of branches. The distributions of these feature values are calculated online, constituting morphometric monitoring of the pseudohyphae population. By providing representative data, the proposed system can improve the effectiveness of morphological characterization, which in turn can help to improve the understanding and control of bioprocesses in which pseudohyphal-like morphologies are found.

Online monitoring of the morphology of an industrial sugarcane biofuel yeast strain via in situ microscopy.

In industrial yeast fermentation processes, single-cell yeast suspensions are usually preferable to cells in aggregates, as single cells exhibit a larger contact area with the nutrient medium, which in many cases helps optimize the process. In addition to affecting fermentation time and efficiency, cell aggregates (e.g., pseudohyphal yeast morphology) may also impair centrifugation systems, one of the most expensive and complex steps of the production process that involves the recovery of yeast cells for subsequent fermentation cycles. To date, no standard technique allows for a systematic diagnosis of yeast morphology in real time during sugarcane biofuel fermentation. Accordingly, we investigate an in situ microscope (ISM) for online monitoring of the density and morphology of an industrial Saccharomyces cerevisiae strain widely used in Brazilian distilleries (PE-2). During batch and repeated batch sugarcane molasses fermentation, the instrument revealed single cells, budding yeast cells, and pseudohyphae, all in a variety of sizes and shapes. The ISM image analysis indicated that the volume of single yeast cells increased by roughly 40% over the lag phase before budding and remained approximately constant thereafter. Pseudohyphae with three and more cells appeared mostly during the stationary phase. Cooling problems were simulated by raising the temperature from 33 to 45 °C. During this thermal stress, single cells as well as budding cells and pseudohyphae forming cells became smaller and exhibited intracellular inhomogeneities. From these results, we conclude that an ISM is a useful tool for monitoring yeast morphology during sugarcane fermentation. Atypical morphologies can be detected early and be used as an automatic warning system.