A viable cell count is essential to evaluate the kinetics of cell growth. Since the hemocytometer was first used for counting blood cells, several variants of the methodology have been developed towards reducing the time of analysis and improving accuracy through automation of both sample preparation and counting. The successful implementation of automated techniques relies in the adjustment of cell staining, image display parameters and cell morphology to obtain equivalent precision, accuracy and linearity with respect to the hemocytometer. In this study we conducted the validation of three trypan blue exclusion-based methods: manual, semi-automated, and fully automated; which were used for the estimation of density and viability of cells employed for the biosynthesis and bioassays of recombinant proteins. Our results showed that the evaluated attributes remained within the same range for the automated methods with respect to the manual, providing an efficient alternative for analyzing a huge number of samples.

The temperature inducible expression system, based on the pL and/or pR phage lambda promoters regulated by the thermolabile cI857 repressor has been widely use to produce recombinant proteins in prokariotic cells. In this expression system, induction of heterologous protein is achieved by increasing the culture temperature, generally above 37°C. Concomitant to the overexpression of heterologous protein, the increase in temperature also causes a variety of complex stress responses. Many studies have reported the use of such temperature inducible expression system, however only few discuss the simultaneous stress effects caused by recombinant protein production and the up-shift in temperature. Understanding the integral effect of such responses should be useful to develop improved strategies for high yield protein production and recovery. Here, we describe the current status of the heat inducible expression system based on the pL and/or pR λ phage promoters, focusing on recent developments on expression vehicles, the stress responses at the molecular and physiological level that occur after heat induction, and bioprocessing factors that affect protein overexpression, including culture operation variables and induction strategies.

Based on partial 16S sequences, we previously described a novel group of nonsymbiotic, acetylene reduction activity-positive actinomycetes which were isolated from surface-sterilized roots of Casuarina equisetifolia growing in Mexico. An amplified rRNA restriction analysis confirmed that these actinomycetes are distinct from Frankia , a finding substantiated by a 16S rRNA gene phylogenetic analysis of two of the Mexican isolates. Further support for these actinomycetes being separate from Frankia comes from the very low DNA-DNA homology that was found. Nevertheless, the Mexican isolates may be diazotrophs based not only on their ability to grow in N-free medium and reduce acetylene to ethylene but also on the results from ¹⁵ N isotope dilution analysis and the finding that a nifH gene was PCR amplified. A comparison of the nifH sequences from the various isolates showed that they are closely related to nifH from Frankia ; the similarity was 84 to 98% depending on the host specificity group. An analysis of complete 16S rRNA gene sequences demonstrated that the two strains analyzed in detail are most closely related to actinobacteria in the Thermomonosporaceae and the Micromonosporaceae .

Fusarium solani has drawn phytopathogenic, biotechnological, and medical interest. In humans, it is associated with localized infections, such as onychomycosis and keratomycosis, as well as invasive infections in immunocompromised patients. One pathogenicity factor of filamentous fungi is biofilm formation. There is still only scarce information about the in vitro mechanism of the formation and composition of F. solani biofilm. In this work, we describe the biofilm formed by a clinical keratomycosis isolate in terms of its development, composition and susceptibility to different antifungals and ultraviolet light (UV) at different biofilm formation stages. We found five biofilm formation stages using scanning electron microscopy: adherence, germination, hyphal development, maturation, and cell detachment. Using epifluorescence microscopy with specific fluorochromes, it was elucidated that the extracellular matrix consists of carbohydrates, proteins, and extracellular DNA. Specific inhibitors for these molecules showed significant biofilm reductions. The antifungal susceptibility against natamycin, voriconazole, caspofungin, and amphotericin B was evaluated by metabolic activity and crystal violet assay, with the F. solani biofilm preformation to 24 h increased in resistance to natamycin, voriconazole, and caspofungin, while the biofilm preformation to 48 h increased in resistance to amphotericin B. The preformed biofilm at 24 h protected and reduced UV light mortality. F. solani isolate could produce a highly structured extra biofilm; its cellular matrix consists of carbohydrate polymers, proteins, and eDNA. Biofilm confers antifungal resistance and decreases its susceptibility to UV light. The fungal biofilm functions as a survival strategy against antifungals and environmental factors.