New chitosan Schiff base and its nanocomposite: Removal of methyl green from aqueous solution and its antibacterial activities.

New chitosan Schiff base (CS-NB) and its CS-NB-NiFe nanocomposite have been prepared and characterized by FTIR spectroscopy, XRD, SEM and DSC. FT-IR spectra and XRD patterns revealed the preparation of chitosan Schiff base CS-NB and its CS-NB-NiFe nanocomposite. DSC demonstrated the endo and exothermic correspondence the evaporation of solvent and decomposition of pyranose ring, respectively. Antibacterial activities was evaluated for the as-prepared compounds against two Gram-positive (Staphylococcus aureus and Bacillus cereus) and two Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria and the results shows that the antibacterial activities of the compounds are found to be stronger than that of chitosan. The order of antibacterial effect according to inhibitory zone around is as follows: S. aureus > E. coli > B. cereus > P. aeruginosa. In addition, the removal of methyl green (MG) dye using CS-NB and its CS-NB-NiFe nanocomposite were analyzed and results showed that the compounds can be effectively used to remove of MG from aqueous solution. Results show that the percentage removal of MG by nanocomposite is higher than Schiff base.

A flow cytometric cell-cycle assay using methyl green.

Methyl green (MG), a conventional, low-cost histological stain, was used to design a flow cytometric cell-cycle/DNA-ploidy assay. On fluorometry, MG absorbed maximally at 633-nm, showed negligible fluorescence in free-state, but emitted brightly when bound to DNA. Optimal dye and cell concentrations for staining and effects of time and photobleaching on stained cells were determined for a lyse-permeabilize-stain protocol. Linearity of DNA-binding, coefficients-of-variation of G0/G1-peaks and minimal carryover were confirmed. Assay results correlated highly with a propidium iodide-based kit in 29 acute lymphoblastic leukemia specimens. The MG-based DNA-ploidy assay represented an accurate and inexpensive alternative to conventional PI-based assays.

High-throughput quantitative histology in systemic sclerosis skin disease using computer vision.

BACKGROUND: Skin fibrosis is the clinical hallmark of systemic sclerosis (SSc), where collagen deposition and remodeling of the dermis occur over time. The most widely used outcome measure in SSc clinical trials is the modified Rodnan skin score (mRSS), which is a semi-quantitative assessment of skin stiffness at seventeen body sites. However, the mRSS is confounded by obesity, edema, and high inter-rater variability. In order to develop a new histopathological outcome measure for SSc, we applied a computer vision technology called a deep neural network (DNN) to stained sections of SSc skin. We tested the hypotheses that DNN analysis could reliably assess mRSS and discriminate SSc from normal skin. METHODS: We analyzed biopsies from two independent (primary and secondary) cohorts. One investigator performed mRSS assessments and forearm biopsies, and trichrome-stained biopsy sections were photomicrographed. We used the AlexNet DNN to generate a numerical signature of 4096 quantitative image features (QIFs) for 100 randomly selected dermal image patches/biopsy. In the primary cohort, we used principal components analysis (PCA) to summarize the QIFs into a Biopsy Score for comparison with mRSS. In the secondary cohort, using QIF signatures as the input, we fit a logistic regression model to discriminate between SSc vs. control biopsy, and a linear regression model to estimate mRSS, yielding Diagnostic Scores and Fibrosis Scores, respectively. We determined the correlation between Fibrosis Scores and the published Scleroderma Skin Severity Score (4S) and between Fibrosis Scores and longitudinal changes in mRSS on a per patient basis. RESULTS: In the primary cohort (n = 6, 26 SSc biopsies), Biopsy Scores significantly correlated with mRSS (R = 0.55, p = 0.01). In the secondary cohort (n = 60 SSc and 16 controls, 164 biopsies; divided into 70% training and 30% test sets), the Diagnostic Score was significantly associated with SSc-status (misclassification rate = 1.9% [training], 6.6% [test]), and the Fibrosis Score significantly correlated with mRSS (R = 0.70 [training], 0.55 [test]). The DNN-derived Fibrosis Score significantly correlated with 4S (R = 0.69, p = 3 × 10- 17). CONCLUSIONS: DNN analysis of SSc biopsies is an unbiased, quantitative, and reproducible outcome that is associated with validated SSc outcomes.

A dual surface inorganic molecularly imprinted Bi2WO6-CuO/Ag2O heterostructure with enhanced activity-selectivity towards the photocatalytic degradation of target contaminantst.

In this work, a high-surface-area dual inorganic molecularly imprinted (DIMI) Bi2WO6/CuO/Ag2O photo-catalyst was developed for the selective photocatalytic degradation of methyl green (MG) and auramine O (AO) dyes as target pollutants. The DIMI-Bi2WO6/CuO/Ag2O heterojunction was synthesized by a sono-chemically assisted sol-gel method by coating a layer of molecularly imprinted Ag2O/CuO on the surface of Bi2WO6 nanocubes with MG and AO as the templates. This was followed by calcination for the removal of target molecules and annealing for Ag/Cu oxide preparation. This novel photocatalyst was prepared to overcome the challenge of the co-existing non-target molecules, which has limited the photocatalytic degradation performance. The surface DIMI sites could act as surface defects for accelerating the separation of photogenerated holes and electrons, which led to the increased generation of OH radicals. Moreover, the DIMI sites had increased binding affinity toward MG and AO via the formation of multiple H bonds and electrostatic bonds, which were confirmed by FTIR spectroscopy, PL and EIS studies. The surface DIMI sites led to the increased adsorption and improved local concentration of MG and AO on Bi2WO6/CuO/Ag2O. Consequently, the heterojunction properties of the final DIMI product accelerated the transfer and separation of photogenerated carriers. The high binding affinity of the DIMI sites to MG and AO confirmed the selective recognition, which was tested in the presence of coexisting pollutant dyes. The other characterizations confirmed the successful fabrication and high photocatalytic activity of the high-surface-area DIMI-Bi2WO6/CuO/Ag2O heterostructured composite. In general, the superior interfacial electronic interactions, high migration efficiency of photoinduced charge carriers, and strong visible light absorption of the prepared photocatalyst resulted in good photocatalytic performance.

A simple morphometric analysis method for dermal microstructure using color thresholding and moments.

BACKGROUND: Proper assessment of dermal collagen fibers by dermatologists and researchers is essential. Histologic evaluation methods have limitations. We present a simple method for measurement of collagen fibers in human skin using Masson's trichrome staining. MATERIALS AND METHODS: Normal skin specimens from a cadaver were processed with Masson's trichrome, which can effectively stain collagen fibers blue with aniline dye. Optical photomicrographs of these slides were analyzed using ImageJ software. Color image processing, a histogram-based function of ImageJ for image segmentation, was performed with color moments thresholding technique. We selected blue areas by adjusting the blue channel to include specific values. The selected areas were highlighted and evaluated. We divided the image into layers of 0.09-mm2 areas from the top to bottom of the dermis. Each area was cropped and evaluated. RESULTS: Quantitative assessment yielded the quantitative size occupied by collagen fibers in an area of 0.09 mm2 . Calculation of the percentage in each area can be used to determine the density of collagen fibers. CONCLUSION: Measurements obtained with our method can be applied to research on dermal collagen fibers. We present a convenient quantitative assessment method for the dermal constituents in Masson's trichrome-stained slides.

The Diagnostic Role of Methyl Green-Pyronin Y Staining in Oral Leukoplakia and Oral Squamous Cell Carcinoma: An Exfoliative Cytology-Based Cytomorphometric Analysis.

BACKGROUND: Oral exfoliative cytology is a noninvasive and nonpainful technique for early diagnosis of oral potentially malignant disorders and oral cancer, and the use of cytomorphometry ameliorates its diagnostic reliability. The objective of the present study was to analyze methyl green-pyronin Y (MGP)-stained oral exfoliated cells (OECs) of oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) by cytomorphometry. MATERIALS AND METHOD: An observational study was conducted on 150 individuals equally divided into three groups: normal mucosa, OL, and OSCC. Smears were prepared from OECs and stained with MGP. Cytomorphometry was done for 100 cells per subject, and various cell and nuclear parameters were measured and calculated. RESULTS: The Kruskal-Wallis test with post hoc correlation showed significant differences in nucleus and cell diameter (ND, CD), nucleus and cell area (NA, CA), nucleus and cell perimeter (NP, CP), and nucleus to cytoplasmic (N:C) ratio for diameter, perimeter, and area. Spearman's ρ correlation of various N:C ratio methods showed good correlation between N:C perimeter and diameter ratio, N:C diameter and ellipse ratio, and N:C area and ellipse ratio. Additional morphological factors showed significant relations for both cell and nuclear regularity factor, shape factor, and nuclear contour index. DISCUSSION: MGP-based cytomorphometry showed a significant decrease in CD, CA, and CP and increase in ND, NA, NP, and N:C ratio from normal mucosa to OL and OSCC. MGP proved its worth as an effective stain for OECs, despite its strict standardization.

Alginate/PAMAM dendrimer - Halloysite beads for removal of cationic and anionic dyes.

The alginate beads have been widely used for dye removal. However, most of the systems described in literature refer to one type of dyes and the adsorbents of cationic ones are dominant. In this study, the composite alginate beads were prepared and characterized. The system was obtained by encapsulation of polyamidoamine - functionalized halloysite nanotubes in alginate (Alg/Hal_PAMAM beads). The adsorptive properties of the beads towards model dyes - cationic methyl green (MG) and anionic sunset yellow FCF (SY) - were investigated with reference to pH, adsorbent dose, time, dye concentration and temperature. The adsorption capacities were improved compared to pure alginate beads. The kinetic data were best correlated to the pseudo-second-order model. The intraparticle diffusion model showed two (MG) or one (SY) adsorptive steps of mass transport. The Freundlich isotherm model was suitable for describing the adsorption process thus the composite beads were characterized by heterogeneous sites. The thermodynamic parameters indicated the spontaneous and endothermic nature of the adsorption process. It was also found that the process was favorable. Additionally, the beads could be reused with satisfactory removal efficiency in several cycles. Therefore, Alg/Hal_PAMAM beads could be considered as promising material for simultaneous removal of cationic and anionic dyes.

Evaluation of photocatalytic fuel cell (PFC) for electricity production and simultaneous degradation of methyl green in synthetic and real greywater effluents.

Recycling of alternative water sources particularly greywater and recovery of energy from wastewater are gaining momentum due to clean water scarcity and energy crisis. In this study, the photocatalytic fuel cell (PFC) employing ZnO/Zn photoanode and CuO/Cu photocathode was successfully designed for effective greywater recycling as well as energy recovery. The photoelectrodes were analyzed using X-ray diffraction (XRD), field-emission scanning electron microscopy (FESEM), energy dispersive X-ray (EDX) and fourier transform infrared (FTIR) spectroscopy. The PFC performance in terms of electricity generation and parallel methyl green (MG) degradation were evaluated under operating parameters such as electrolyte type, initial MG concentration and solution pH. The results showed that the addition of Na2SO4 electrolyte, MG concentration of 40 mg L-1 and solution pH of 5.2 improved the short circuit current density (Jsc) and power density (Pmax) in the as-constructed PFC. Such a system also afforded highest MG and chemical oxygen demand (COD) removal efficiencies after 4 h of irradiation. The photoanodes used in this study demonstrated great recyclability after four repetition tests. The COD removal was reduced to some extents when the PFC treatment was tested in the real greywater under optimal conditions. Various greywater quality parameters including ammoniacal nitrogen (NH3-N), turbidity, pH and biochemical oxygen demand (BOD5) were also monitored. The phytotoxicity experiments via Vigna radiate seeds indicated a reduction in the phytotoxicity.

DNA staining in agarose and polyacrylamide gels by methyl green.

Methyl green (MG) is an inexpensive, nonproprietary, traditional histological stain for cell nuclei. When bound to DNA and upon excitation with orange-red light, it fluoresces brightly in the far red region. We compared MG with ethidium bromide (EtBr), the conventional stain for DNA in gels, and Serva DNA stain G™ (SDsG), a proprietary stain marketed as a safer alternative to EtBr for staining of electrophoresed DNA bands in agarose and polyacrylamide gels. DNA-MG fluorescence was recorded and 2.4 µg/ml MG produced crisp images of electrophoresed DNA after incubation for 10 min. Stain solutions were stable and detection limits for faint bands as well as relative densitometric quantitation were equivalent to EtBr. MG, EtBr and SDsG cost 0.0192, 0.024 and 157.5 US cents/test, respectively. MG is an effective stain for visualizing DNA in agarose and polyacrylamide gels. Its major advantages including low cost, comparable quality of staining, storage at room temperature, photo-resistance and low mutagenic profile outweigh its disadvantages such as staining of tracking dye and requirement for a gel documentation system with a red filter.

Enzyme-linked immunosorbent assay for group A Streptococcal anti-DNase B in human sera, using recombinant proteins - Comparison to the DNA methyl green micromethod.

Among the four known Streptococcal nucleases comprising of DNase A, B, C and D; DNase B is the most common, and determination of the levels of antibody to DNase B (ADB) is often used to confirm a clinical diagnosis of Streptococcus pyogenes/group A Streptococcal (GAS) infection. The commonly used assays for antibodies that neutralize DNase B or streptolysin O activity use partially purified antigens that often fail to detect antibody changes subsequent to culture documented infections. Therefore, an enzyme-linked immunosorbent assay (ELISA) was developed employing his-tagged recombinant DNase B as plate antigen for comparison to the commonly used DNA methyl green micromethod (DMGM). DNAs from various Streptococcal species were screened for presence of dnaseB gene by PCR. Measurements of ADB in sera collected from subjects belonging to different ages, and ethnic groups were used to compare the two methods. dnaseB was not detected by PCR in DNA samples isolated from different strains of group B (GBS), C (GCS) and G (GGS) Streptococci. The ADB based ELISA proved to be highly sensitive and more responsive to changes in antibody concentration than DMGM. Use of recombinant DNase B eliminates the variability associated with the enzyme, partially purified from Streptococcal culture supernatants from various commercial sources and may provide a more reliable source of antigen to a wider group of laboratories concerned with GAS diagnosis.

A rapid nuclear staining test using cationic dyes contributes to efficient STR analysis of telogen hair roots.

Telogen hairs presented in the crime scene are commonly encountered as trace evidence. However, short tandem repeat (STR) profiling of the hairs currently have low and limited use due to poor success rate. To increase the success rate of STR profiling of telogen hairs, we developed a rapid and cost-effective method to estimate the number of nuclei in the hair roots. Five cationic dyes, Methyl green (MG), Harris hematoxylin (HH), Methylene blue (MB), Toluidine blue (TB), and Safranin O (SO) were evaluated in this study. We conducted a screening test based on microscopy and the percentage of loss with nuclear DNA, in order to select the best dye. MG was selected based on its specific nuclei staining and low adverse effect on the hair-associated nuclear DNA. We examined 330 scalp and 100 pubic telogen hairs with MG. Stained hairs were classified into five groups and analyzed by STR. The fast staining method revealed 70% (head hair) and 33.4% (pubic hair) of full (30 alleles) and high partial (18-29 alleles) STR profiling proportion from the lowest nuclei count group (one to ten nuclei). The results of this study demonstrated a rapid, specific, nondestructive, and high yield DNA profiling method applicable for screening telogen hairs.

Application of the DNA-specific stain methyl green in the fluorescent labeling of embryos.

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored until recently. In this article, we illustrate the method for preparing a methyl green aqueous stock solution, that when diluted can be used as a very convenient fluorescent nuclear label for fixed cells and tissues. Easy procedures to label whole zebrafish and chick embryos are detailed, and examples of images obtained shown. Methyl green is maximally excited by red light, at 633 nm, and emits with a relatively sharp spectrum that peaks at 677 nm. It is very inexpensive, non-toxic, highly stable in solution and very resistant to photobleaching when bound to DNA. Its red emission allows for unaltered high resolution scanning confocal imaging of nuclei in thick specimens. Finally, this methyl green staining protocol is compatible with other cell staining procedures, such as antibody labeling, or actin filaments labeling with fluorophore-conjugated phalloidin.

A histochemical comparison of methyl green-pyronin, and hematoxylin and eosin for detecting apoptotic cells in oral squamous cell carcinoma, oral leukoplakia, oral submucous fibrosis and normal oral mucosa.

Analysis of apoptotic cells in oral pathological states could be useful for determining the rates of tissue turnover, which would help determine prognosis. The use of histochemical stains such as hematoxylin and eosin (H & E) and methyl green-pyronin (MGP) can provide a simple and cost-effective method for detecting apoptotic cells. We compared the efficacy of MGP and H & E for detecting apoptotic cells in oral squamous cell carcinoma (OSCC), oral leukoplakia (OL), oral submucous fibrosis (OSMF) and normal oral mucosa (NOM). Ten cases each of OSCC, OSMF, OL and NOM were retrieved from the archives and two serial sections were stained, one with H & E and the other with MGP. Apoptotic cells were identified at 100 x magnification and the apoptotic index was calculated. Apoptotic cells were distinguished more readily in MGP stained sections than in those stained with H & E. Also, the apoptotic cell count was greater in OSCC compared to OL, OSMF and NOM. We concluded that MGP staining can be used as a routine, cost-effective method for detecting apoptotic cells.

A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green.

The increasing need for multiple-labeling of cells and whole organisms for fluorescence microscopy has led to the development of hundreds of fluorophores that either directly recognize target molecules or organelles, or are attached to antibodies or other molecular probes. DNA labeling is essential to study nuclear-chromosomal structure, as well as for gel staining, but also as a usual counterstain in immunofluorescence, FISH or cytometry. However, there are currently few reliable red to far-red-emitting DNA stains that can be used. We describe herein an extremely simple, inexpensive and robust method for DNA labeling of cells and electrophoretic gels using the very well-known histological stain methyl green (MG). MG used in very low concentrations at physiological pH proved to have relatively narrow excitation and emission spectra, with peaks at 633 and 677 nm, respectively, and a very high resistance to photobleaching. It can be used in combination with other common DNA stains or antibodies without any visible interference or bleed-through. In electrophoretic gels, MG also labeled DNA in a similar way to ethidium bromide, but, as expected, it did not label RNA. Moreover, we show here that MG fluorescence can be used as a stain for direct measuring of viability by both microscopy and flow cytometry, with full correlation to ethidium bromide staining. MG is thus a very convenient alternative to currently used red-emitting DNA stains.

Adsorption behavior of light green anionic dye using cationic surfactant-modified wheat straw in batch and column mode.

An agricultural by-product, natural wheat straw (NWS), was soaked in 1 % cationic surfactant (hexadecylpyridinium bromide, CPB) solution for 24 h (at 293 K), and modified wheat straw (MWS) was obtained. Analysis of FTIR, XFR, and nitrogen element showed that CPB was adsorbed onto surface of NWS. Then, MWS was used as adsorbent for the removal of light green dye (LG, anionic dye) from aqueous solution. The experiment was performed in batch and column mode at room temperature (293 K). Sodium chloride (up to 0.1 mol/L) existed in solution was not favor of LG dye adsorption. The equilibrium data were better described by Langmuir isotherm, and adsorption capacity of q m from Langmuir model was 70.01 ± 3.39 mg/g. In fixed-bed column adsorption mode, the effects of initial LG concentration (30, 50, 70 mg/L) and flow rate (6.5, 9.0, 14.5 mL/min) on adsorption were presented. Thomas and modified dose-response models were used to predict the breakthrough curves using nonlinear analysis method, and both models can fit the breakthrough curves. Theoretical and experimental breakthrough curves were drawn and compared. The results implied that MWS can be used as adsorbent material to remove LG from aqueous solution.

The role of Phe329 in binding of cationic triarylmethane dyes to human butyrylcholinesterase.

Cationic triarylmethane dyes (TAM(+))s which are used as colorants in industry and as frequent tools and reagents in analytical, cell biological and biomedical research have been recently characterized as reversible inhibitors of human butyrylcholinesterase. In this study, the inhibitory effects of two TAM(+)s, malachite green (MG) and methyl green (MeG) on five human BChE mutants (A277V, P285L, H77L, A328F and F329A) were studied spectrophotometrically at 25°C in 50mM MOPS buffer pH 8, using butyrylthiocholine as substrate. The kinetic results obtained with mutant enzymes were compared to those obtained with recombinant wild type BChE. MG and MeG were found to act as competitive/linear mixed inhibitors of recombinant wild type BChE and all BChE mutants except the F329A mutant. Both dyes caused complex nonlinear inhibition of F329A mutant, pointing to multisite binding. K(i) values for MG and MeG, estimated by nonlinear regression analysis, were 3.8 and 27 µM, respectively, as compared to the 50- to 150-fold lower values observed with recombinant wild type BChE. The observed significant differences in kinetic pattern and K(i) values between recombinant wild type BChE and F329A mutant suggest that phenylalanine at position 329 in human BChE is a critical residue in MG and MeG binding to enzyme.

Controlling of band width, resolution and sample loading by injection system in a simple preparative free-flow electrophoresis with gratis gravity.

In this paper, the controllable band width, resolution and sample loading were investigated by the injection system of free-flow electrophoresis (FFE) with gratis gravity. Two general injection methods were described herein. The first method was the one in which sample injection fluxes were variable with constant background flux, while the second was the one in which the background fluxes were flexible with stable sample flux. With methyl green and crystal violet as two viewable model compounds, a series of experiments were performed, and the experimental results revealed that (1) the sample band width could be under desiring control through the regulation of ratios between sample and background fluxes, (2) the separative resolution could be also adjusted elaborately via the regulation of flux ratios during the separation of methyl green and crystal violet with only one charge disparity, and (3) the sample loading could be conveniently controlled via the flux ratios and an approximate maximum sample loading could be selected under the condition of just completed separation of two adjoining solutes. In addition, it was observed that the flux ratio had soft influence on the separative resolution of two solutes. These results were of significance to the designs on band width, resolution and sample loading in the newly developed FFE device with gratis gravity.

Electrophoretic exclusion for the selective transport of small molecules.

A novel method capable of differentiating and concentrating small molecules in bulk solution termed "electrophoretic exclusion" is described and experimentally investigated. In this technique, the hydrodynamic flow of the system is countered by the electrophoretic velocity to prevent a species from entering into the channel. The separation can be controlled by changing the flow rate or applied electric field in order to exclude certain species selectively while allowing others to pass through the capillary. Proof of principle studies employed a flow injection regime of the method and examined the exclusion of Methyl Violet dye in the presence of a neutral species. Methyl Violet was concentrated almost 40 times the background concentration in 30 s using 6 kV. Additionally, a threshold voltage necessary for exclusion was determined. The establishment of a threshold voltage enabled the differentiation of two similar cationic species: Methyl Green and Neutral Red.

Modifications of the lactate dehydrogenase assay, a histochemical determinant of osteocyte viability--a qualitative study.

We have refined a technique for assessment of osteocyte viability in a canine and murine model using a modification of the lactate dehydrogenase assay (LDH). With this method, viable osteocytes react to form non-reversible tetrazolium-formazan granules, while non-viable osteocytes are distinguished by a methyl green stain. LDH assay in canine and murine models have not been reported and our initial efforts were not successful. We examined the effect of (a) concentration of coenzyme and tetrazole (b) bone specimen thickness (c) ability to use frozen sections and (d) incubation time/dilution. We concluded that a 1000-fold increase in the concentration of coenzyme and tetrazole were required. Fresh bone produced optimal results and near-complete viability. Special considerations must be taken with smaller, more fragile specimens (e.g., mouse bone), such as increasing specimen thickness, dilution of incubation medium and/or the reduction of incubation time. Sections from thawed frozen bone resulted in a diffuse reaction. Osteocyte viability can be assessed via LDH assay in both dog and mouse bones; however, this approach requires modifications from the previous published method.

Histomorphometric analysis of the irides of dogs, camels, buffalos and donkeys.

The present study was carried out to investigate the morphological and histomorphometrical characters of irides in dogs, camels, buffalos, and donkeys. The findings of the study revealed that, morphologically, the irides were consisted of an anterior border layer, a middle layer of connective tissue stroma and a posterior layer of pigmented epithelium. Interestingly, the anterior borders of all investigated animals were not enveloped by a distinct epithelial or endothelial lining, but on contrary, the posterior border was covered by pigmented epithelium. The constrictor and dilator iridial muscles were well developed in dogs, weakly developed in donkeys, and with an intermediate position in camels and buffalos. Morphometric analysis revealed significant species differences in the mean total thickness of the iris and its different layers. In addition significant differences were also found between the ratio of the means of different layers to the total thickness of the iris at the pupillary, middle and ciliary borders. In conclusion, these variations might be related to the different lifestyles and visual behaviour of the investigated animals.