EVALUATION OF DIFFERENT INCUBATION TIMES OF XTT TETRAZOLIUM ASSAY TO ASSESS THE METABOLIC ACTIVITY OF PLANKTONIC YEASTS.

COLETTI, T.M.S.F.A.1*, PAVARINA, A.C.2, DOVIGO, L.N.2, MACHADO, A. L. 2, GIAMPAOLO, E. T. 2, BRUNETTI, I.L. 1

1*_{Depto. de Análises Clínicas, Universidade Estadual Paulista “Julio de Mesquita Filho” Unesp - Av.} Expedicionários do Brasil nο1621, Centro. Araraquara – SP. *tatistarck@ig.com.br

2_{Depto. de Materiais Odontológicos e Prótese, Universidade Estadual Paulista “Julio de Mesquita Filho”}

Keywords: Candida, Metabolism, Quantitative Analysis

Introduction: Yeast infections are an increasingly important problem, particularly in immunocompromised patients. Colorimetric assays of cellular viability are important tools in the study of eukaryotic cell activity and antifungal resistance. Some tetrazolium salts are widely used as indicators of cellular metabolism. XTT, 2,3-bis(2-methoxy-4-nitro-5- sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide has been used to determine the viability of Candida biofilms after antifungal therapies. This method seems to be useful for comparisons involving one strain, but its use may be difficult in attempts to compare different fungal species, especially in the planktonic form. This study performed a comparative assessment of XTT metabolism among differents Candida species in the planktonic form. In addition, different times of XTT incubation were also evaluated. Methods: The microorganism used in this study was an American Type Culture Collection (ATCC; Rockville, Md, USA) strain of C. albicans (90028), C. glabrata (2001), C.

dubliniensis (7987), C. tropicalis (4563) and C. krusei (6258). To obtain the standardized

planktonic suspensions of Candida, the yeasts were individually inoculated in 5 mL of Tryptic Soy Broth and incubated (24h/37°C). Each culture was harvested after centrifugation at 2000 rpm for 10 min, washed twice with sterile distilled water and resuspended in PBS to a turbidity 106 cells ml-1 (MicroScan Turbidity Meter, Siemens). Aliquots of 200μl of each Candida standardized suspension were individually transferred to separate wells of a 96-well microtiter plates. The XTT solution was prepared instantly before use by adding 1.5ml of XTT (1mg/ml in sterile saline; Sigma Chemicals, St.Louis, MO) in 300µL of menadione solution (0.4M Minacetone; Sigma Chemicals). Then, 12µL of XTT was added to each well of the microtiter plate. The plates were incubated in dark at 37°C for 1, 2, 3, 4 and 5h. Then 100μL of the reacted XTT salt solution was transferred to a new 96-wells microtiter plate and the cell viability was analyzed by proportional colorimetric changes and light absorbance measured by a microtiter plate reader (Thermo Plate—TP Reader) at 492 nm. The experiments were performed with ten replicates for each experimental condition and data were statistically analyzed using ANOVA and Tukey's test (α=5%). Results: For all strains evaluated, a relationship between incubation time and color development was noted when the incubation duration is no longer than 3h. The incubation times of 2 and 3h promoted the highest values of absorbance for C.

albicans, with the lowest variation coefficient (approximately 8%). For C. glabrata, C. dubliniensis, C. tropicalis and C. krusei the incubation during 3h was the most effective in

promoting colorimetric changes without an elevated variation coefficient. Discussion: Several investigations already showed that the XTT salt produces color changes that can reflect the number of viable cells in the yeast sample. In addition, some studies suggested a linear relationship between incubation time and color development. Our results are in accordance with the findings from these previous studies. In conclusion, the XTT assay can be considered a valuable tool for examining the behavior of Candida species in planktonic form.

Evaluation of Helminth Eggs and Protozoan Cysts from effluent in a System of Sewage Treatment in Cuiabá / MT.

LIMA, M.A.B.S.1, CARVALHO1, T.S., GOLIN1, R. F., LIMA, Z.M. 1, LIMA, J.B. 1

1* _{Departament of Sanitary and Environmental Engineering}

. Federal University of Mato Grosso. Av. Fernando Corrêa da Costa, s/n°, Coxipó. Cuiabá- MT. *taissalima@msn.com

Keywords: Helminths, Protozoa, Effluent, Reuse

The treated effluent of stabilization ponds can be reused for various purposes, including landscape irrigation and fish farming. However, for the reuse not offer a risk to public health it is necessary that the effluent produce certain microbiological standards. This study analyzed samples of the effluent from the Sewage Treatment named Lagoa Encantada (Cuiabá - Mato Grosso) in order to evaluate the efficiency of the removal of helminth eggs and protozoan cysts; determining the predominant parasite species and also final effluent quality for reuse in landscape irrigation and fish farming, according to the standards analyzed. The samples were collected at Sewage Treatment named Lagoa Encantada (Cuiabá – MT) in the period from november 2009 to november 2010. The samples were analyzed from the effluent entry (stabilization pond), the effluent treatment and treated effluent (station exit). The materials used were: 10 ml pipettes, Pasteur pipettes, 1000 ml beakers, buckets, hose thin, Vortex, centrifuge tubes 50ml, optical microscope, 5l bottles, plates, coverslips, McMaster chamber, solution of formaldehyde, solution aceto acetic acid, ether solution, Lugol solution and zinc sulfate solution. The laboratory technique used for the identification of protozoan cysts was the method of Ritchie (1948) modified (AYRES & MARA, 1996). For the quantification of helminth eggs were used the method Bailenger (1979) modified (WHO, 1989, 2006). We found 06 (six) eggs per liter in the effluent input. Output in the effluent (treated wastewater) were not found helminth eggs or protozoan cysts, indicating that 100% removal of these parasites in the effluent of sewage treatment studied. The helminth eggs found in raw wastewater were predominantly of the genera Ancylostoma, Ascaris, Trichuris and Hymenolepis. The cysts of protozoa were identified as the genera Entamoeba and Giardia. According to WHO, the treated effluent to be used in fish farming, landscape irrigation should not have helminth eggs and protozoan cysts. Thus, the results show that the effluent from the Sewage Treatment named Lagoa Encantada is suited to this kind of reuse with respect to their efficiency in removal of helminth eggs and protozoan cysts, however it is important to consider other microbiological parameters, physicists and chemists.

Financial support: Project Centro de Referencia de Reuso de Água - CRRA/Petrobras/ UFMT

longisetula. A PHYTOPATOTHOGENIC FUNGUS IN STRAWBERRY