Photometric Application of the Gram Stain Method To Characterize Natural Bacterial Populations in Aquatic Environments

The Gram stain method was applied to the photometric characterization of aquatic bacterial populations with a charge-coupled device camera and an image analyzer. Escherichia coli and Bacillus subtilis were used as standards of typical gram-negative and gram-positive bacteria, respectively. A mounting agent to obtain clear images of Gram-stained bacteria on Nuclepore membrane filters was developed. The bacterial stainability by the Gram stain was indicated by the Gram stain index (GSI), which was applicable not only to the dichotomous classification of bacteria but also to the characterization of cell wall structure. The GSI spectra of natural bacterial populations in water with various levels of eutrophication showed a distinct profile, suggesting possible staining specificity that indicates the presence of a particular bacterial population in the aquatic environment.Gram’s method is the most important and fundamental orthodox method for bacterial identification. It classifies bacteria into two groups, gram-negative and gram-positive. The mechanism of Gram staining is based on the fundamental structural and chemical attributes of bacterial cell walls. The cell walls of gram-positive bacteria have a high percentage of peptidoglycan, while those of gram-negative bacteria have only a thin peptidoglycan layer (1–3, 6). In Gram’s method, an insoluble dye-iodine complex is formed inside bacterial cells and is extracted by alcohol from gram-negative but not gram-positive bacteria (6, 12, 16). There are taxonomically gram-variable species, but some cells of gram-negative or gram-positive species may show gram-variable characteristics due to environmental stress, such as unsuitable nutrients, temperature, pH, or electrolytes (3).Functional differences between gram-positive and gram-negative cell walls have been studied with special emphasis on nutrient uptake from the ambient environment. Gram-negative bacteria have a periplasmic space between the lipopolysaccharide layer and the plasma membrane. In this space, binding proteins initially attach to nutrients and take them to a membrane carrier. Gram-positive bacteria lack the periplasmic space and are believed to have no binding proteins (9). Therefore, nutrient uptake from the environment is easier for gram-negative bacteria than for gram-positive bacteria. Because of this difference, the population density of gram-negative bacteria in more oligotrophic environments could be higher than that of gram-positive bacteria (20).Gram staining is commonly used only to reflect cell wall structure. If Gram staining characterizes not only simple taxonomical dichotomy but also multiple biological functions, it may also be used to correlate bacterial cell wall structure with related physiological responses to the environment. In particular, Gram staining could supply ecological information on natural bacterial populations that are difficult to culture by the present technology.Membrane filter methods are widely used for microscopy in aquatic microbiology because of the low population densities of bacteria in many aquatic environments (4, 11, 16). However, these methods sometimes have problems associated with microscopic observations, causing unclear images of bacterial cells on Nuclepore filters when used with the conventional mounting medium (immersion oil; refractive index [nd] = 1.514). Hence, a suitable mounting agent must be applied to obtain precise image analyses of Gram-stained bacteria on Nuclepore filters.In this study, we have established a distinct method to characterize photometric Gram stain images; it involves the Gram stain index (GSI) for specifying natural bacterial populations in various aquatic environments. For this purpose, we have standardized the GSI of typical gram-negative and gram-positive bacteria by using Escherichia coli and Bacillus subtilis, respectively, and compared these GSI values to those of natural bacterial populations of several freshwater environments. The natural waters we investigated were Hyoutaro-ike pond, Matsumi-ike bog, and Lake Kasumigaura, which are oligotrophic, mesotrophic, and eutrophic water bodies, respectively, as previously determined (8, 10, 13, 18, 22, 23).     

Fatty acids from seeds of Pinus pinea L.: composition and population profiling

Pinus pinea L. is widely disseminated all over the Mediterranean Basin. Qualitatively, P. pinea fatty acid seed composition is identical and typical of the genus Pinus. This composition is made of unsaturated oil with several unusual polymethylene-interrupted unsaturated fatty acids. Linoleic acid is the major fatty acid followed by oleic, palmitic and stearic acids. Quantitatively, for all Mediterranean populations, total amounts of fatty acids seem to be fairly constant and independent from their origin. When applying principal component analysis, it seems that there is not a distinct geographical variability. Tunisian populations appear to be integral part of the Mediterranean populations without any particular structuring. Taking into account this research and the data reported in the literature, we can confirm that P. pinea expresses no significant variability. This low genetic diversity revealed by fatty acid composition can be explained by anthropogenetic diffusion of genetically homogeneous reproductive material as early as the first explorations.     

Assessment of Some Heavy Metals in the Dead Sea Mud and Treatment Optimization

This paper describes the experimental remediation of the Dead Sea mud and the quantitative determination of some heavy metals. Herein, two chelating agents were employed as extracting aqueous solution: ethylenediaminetetraacetic acid (EDTA) and citric acid. The study focused on the main known heavy metals that were reported previously to be in the Dead Sea mud, which are Co, Ni, Pb, Zn, and Cr. Findings had indicated that citric acid was efficient in the removal of the aforementioned heavy metals. Physicochemical parameters that were expected to affect the removal of metals in the Dead Sea mud were optimized. Those parameters were the chelating agent concentration, mixing time and speed, type of washing water, temperature, and pH. The results showed that the best removal of heavy metals from Dead Sea mud can be achieved under optimum citric acid concentration, 1.5 g/50 mL for treatment of 10 g mud. Optimum mixing speed and time were found to be 800 rpm and 1 hr, respectively. Regarding washing water, it was found that the use of the same water for repeated washing provided better removal percentages. pH values and temperature had effect on removal percentages of the heavy metals from mud. However, working at pH 7 and room temperature would provide convenient results for heavy metal removal.     

High doses of ultraviolet-C irradiation increases vasoactive intestinal contractor/endothelin-2 expression in keratinocytes of the newborn mouse epidermis

We examined the expression profiles of vasoactive intestinal contractor/endothelin-2 (VIC/ET-2) at both gene and peptide level in skin irradiated with different ultraviolet wavelengths. We found that VIC/ET-2 gene expression is sensitive only to ultraviolet-C (UVC) irradiation and has an immediate response. These results provide direct evidence that high doses of UVC irradiation induce an increase in gene expression and protein production of VIC/ET-2 and endothelin (ET) receptors in a dose-dependent manner in epidermal keratinocytes. We suggest that VIC/ET-2 can play an essential role in the maintenance, protection and hyperpigmentation of the epidermis exposed to UVC irradiation from artificial or natural sources.