2007 International Nuclear Atlantic Conference - INAC 2007 Santos, SP, Brazil, September 30 to October 5, 2007 ASSOCIAÇÃO BRASILEIRA DE ENERGIA NUCLEAR - ABEN ISBN: 978-85-99141-02-1
ALTERATIONS INDUCED IN ESCHERICHIA COLI CELLS BY GAMMA RADIATION J. Kappke1, H. R. Schelin1, S.A. Paschuk1, V. Denyak1, E. R. da Silva1, E. F. O. de Jesus2, R. T. Lopes2, N. Carlin3 and E. S. Toledo3 Federal University of Technology - Paraná, CPGEI, UTFPR Av. Sete de Setembro, 3165, Curitiba, PR, 80230-901, Brazil
[email protected] [email protected] [email protected]
1
2
LIN-COPPE/UFRJ, Rio de Janeiro, Brazil
[email protected] [email protected]
3
Instituto de Física, USP, São Paulo, Brazil
[email protected]
ABSTRACT Modifications occurred in Escherichia coli cells exposed to gamma radiation (60Co source) were investigated. The irradiations were done at the LIN-COPPE laboratory of the UFRJ and the analysis at the Biology Department of the UTFPR. The E. coli cells were irradiated with 30, 60, 90, 120, 150, 180, 210, 240, 300, 480, 600 e 750 Gy doses. The samples were analyzed with Gram-stain, biochemical tests in EPM, MIO and Lysine Broth, Simmons Cytrate Medium and Rhamnose Broth, antibiogram and isolation of auxotrophic mutants. It was observed that for the received doses the E. coli didn’t show morphological alterations in the tests. Some E. Coli cells showed to be able to deaminade the L-trytophan or they changed their sensibility for amoxillin and cephaloonine after the irradiation. The existence of aauxotrophic mutants after irradiation was also verified.
1. INTRODUCTION Ionizing radiation is able to form ions and free radicals that can react with the DNA and other cell constituents producing mutations that, depending on the kind, could result in the cell’s death. The number of interactions and mutations is proportional to the absorbed dose, but the provoked effects by the interaction of the ionizing radiation with the biological tissues also depend on the kind of radiation and the exposition time, and on the state in which the cell is in its cellular cycle and the oxygen concentration in the radiation environment [1, 2]. The present work intends to verify the occurrence of transformations in E. coli (ATCC 25922) exposed to gamma radiation (60Co source). Morphological alterations in the irradiated cells were checked through optical microscopy and analysis of the bacterial colonies’ cultural characteristics. The action of the irradiated microorganism on the chemical composes through biochemical tests was also checked, verifying the alterations on the biochemical behaviors of the irradiated cells in the comparison with the non-irradiated cells [3, 4].
2. METHODOLOGY 2.1. Sample Preparation The samples were prepared following the diagram showed in Figure 1 [5, 6]. E. coli cultivation in BHI broth for 24 hours.
Centrifugation and ressuspention of the cells in saline solution
Dilution in saline solution.
0.02 mL of the 10-5 dilution were inoculated in MacConkey Agar.
Irradiation
0.85%.
Figure 1: Schematic diagram, showing the procedure for sample preparation with the Gamma Excell irradiator (GC-220E) used in the experiment. 2.2. Irradiation After scattering the aliquots, the bacteria were submitted to gamma radiation (60Co) with doses of 30, 60, 90, 120, 150, 180, 210, 240, 300, 480 and 780 Gy in triplicate. Nonirradiated plates were used as growing standard of E. coli. The irradiator used was the Gamma Excel (GC-220E) from the COPPE-UFRJ, as shown in Figure 2, with a dose rate of ~60Gy/min [5, 6].
Figure 2: The Gamma Excell irradiator (GC-220E) used for the experiment.
2.3 Analysis of Samples 2.3.1. Morphological analysis of the irradiated microorganisms
INAC 2007, Santos, SP, Brazil.
The cultural characteristics of the microorganisms were evaluated concerning the shape (heterogene/homogene), size, structure, texture and pigmentation of the colony and compared to non-irradiated microorganisms. Microscopic examinations were done for classification and shape purposes (Gram staining) [7, 8, 9]. 2.3.2. Metabolic activity analysis The physiological characteristics of the irradiated microorganisms were evaluated through biochemical tests. The cells were initially cultivated in nutrient agar and incubated at 37°C for a period of 24 hours. Aliquots were inoculated in the specific environment medium for each biochemical test. The minikit used in this analysis consisted of a set of five cultural medium environment to identify enterobacteria, which are: EPM Medium, Lysine Broth, MIO Medium, Simmons Citrate Medium and Rhamnose Broth. The functions of each medium of the biochemical tests are shown in Table 1 [10].
Table 1. Minikit Mediums Characteristics Cuture Medium EPM
Function Verifies the occurrence of glucose fermentation through the characteristic color formation in the base of the tube, the capacity to produce sulfidric gas with consequent darkening the base of the tube, Ltriptophan deaminase production with green-moss color in the top of the tube.
Lysine Broth
Determines the enzymatic ability to promote descaboxilation of lysine amino acid, alkalinizing the culture medium.
MIO
Alows reading of the motility though the turbidity of the medium, descaboxilation of the ornitine trough the formation of the yellow coloration in the medium and the production of the indol is evidenced with the addition of the kovacs reagent and a red ring formation.
Simmons Citrate Determines the ability of the microorganism to utilize the citrate as the only source of carbon through the color changes from the medium to blue. Rhamnose Broth The Rhamnose fermentation test verifies if the microorganism is capable or not of fermenting that sugar through the color changes to yellow.
3. RESULTS AND DISCUTION After the irradiation the E. coli showed to be able to survive to doses up to 210Gy being cultivated in Agar MacConkey at 37ºC. In the present work, the E. coli (ATCC 25922) has shown to be sensitive to radiation. When exposed to radiation in food, the necessary dose for a 90% reduction of the E. coli cells is verified with doses around 260 Gy. Probably the INAC 2007, Santos, SP, Brazil.
culture medium (selective MacConkey Agar) utilized for the E. coli affected its sensitivity to the gamma radiation [11]. The characteristics of the E. coli irradiated and cultivated in positive lactose Macconkey Agar showed an intense pink-reddish coloration, dry and opaque with yeast smell, typical characteristics of the E. coli when cultivated in this medium [8, 9]. There were no alterations observed in the microscopic characteristics of the E. coli cells after the irradiation with gamma ray beams. The E. coli before and after irradiation showed themselves as very short rods like as expected. It is the contrary of what was observed for the E. coli exposed to much smaller doses with proton beams and with UV light. In the latter case a cellular elongation was observed in a previous work [8, 9]. When submitted to several biochemical tests the E. coli irradiated with different gamma radiation doses (60Co) showed the characteristic behavior of the non-irradiated E. coli. The only exception was the deamination of L-tryptophan test, through the tryptophan deaminase enzyme [10]. Figure 3 shows E. coli non-irradiated in biochemical tests and Figure 4 shows E. coli irradiated with gamma radiation.
Figure 3: EPM Medium, Lysine Broth, MIO Medium, Simmons Citrate Medium Rhamnose Broth, inoculated with the non-irradiated E. coli.
Figure 4: EPM Medium, Lysine Broth, MIO Medium, Simmons Citrate Medium Rhamnose Broth, inoculated with the irradiated E. coli.
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3. CONCLUSIONS Through the performed analysis it was possible to show that the 240 Gy dose is lethal for the E. coli cultivated in MacConkey Agar. Microscopic morphological alterations were not detected in irradiated E. coli cells and macroscopic alterations in the colonies were not verified either. The performed biochemical tests showed that the irradiated and the non-irradiated E. coli cells had the same behavior in the cultural medium Lysine Broth , MIO Medium, Simmons Citrate Medium and Rhamnose Broth. However, after the irradiation, the E. coli changed its behavior for the EPM Medium, becoming able to deaminade the Ltryptophan. It was possible to verify that the gamma radiation in non-lethal doses showed to be able to provoke alteration in the biochemical behavior of the E. coli.
ACKNOWLEDGMENTS The authors want to thank CNPq, CAPES and Fundação Araucária for financial support of this work. REFERENCES 1. Bernardo, L. C.; De Oliveira, M. B. N.; Da Silva, C. R.; Dantas, F. J. S.; De Mattos, J. C. P.; Caldeira-De-Araújo, A.; Moura, R. S.; Bernardo-Filho, M. Biological “Effects of Rutin on the Survival of Escherichia coli AB1157 and on the Electrophoretic Mobility of Plasmid PUC 9.1 DNA” CELLULAR AND MOLECULAR BIOLOGY TM, v. 48, (2002). 2. Tauhata, L.; Salati, I. P. A.; Di-Prinzio, R. RADIOPROTEÇÃO E DOSIMENTRIA: Fundamentos. Rio de Janeiro: Instituto de Radioproteção e Dosimetria – CNEN (1998). 3. Black, J. G., “Microbiologia: Fundamentos e Perspectivas”. Rio de Janeiro: Editora Guanabara Koogan S.A. (2002). 4. Tortora, G. J.; Funke, B. R.; Case, C. L., “Microbiologia” Porto Alegre: Artmed (2000). 5. Kappke, J., “ESTUDO DOS DANOS PROVOCADOS EM Escherichia coli PELA RADIAÇÃO ELETROMAGNÉTICA”. Trabalho de Conclusão de Curso, Curso Superior de Tecnologia em Radiologia CEFET-PR, 48p, (2004). 6. Kappke, J., Silva, E. R., Schelin, H. R. Paschuk, S. A., Pashchuk A., Oliveira, A., Carlin, N., Szanto, E. M., Takahashi, J., Souza, J. C. “Evaluation of Escherichia coli cells damages induced by ultraviolet and proton beam radiation.” Brazilian Journal of Physics, v.35, n.3b, pp.805-807 (2005). 7. Leitão, M. F. de F.; Hagler, L.C.S.M.; Hagler, A. N.; Menezes, T. J. B., “Tratado de Microbiologia”. São Paulo: Editora Menole Ltda. (1988). 8. Pelczar Jr, M. J.; Chan, E. C. S.; Krieg, N. R., “MICROBIOLOGIA CONCEITOS E APLICAÇÕES”. 2a ed. São Paulo: Editora Mackron Books.( 1996). 9. Ribeiro, M. C.; Soares M. M. S. R., MICROBIOLOGIA PRÁTICA: ROTEIRO E MANUAL – BACTÉRIAS E FUNGOS. 1ª ed. São Paulo: Editora Atheneu (2002). 10. Pilonetto, M. BULÁRIO Newprov. www.newprov.com.br. (2006). 11. Joint FAQ/IAEA/WHO Study Group, HIGH-DOSE IRRADIATION: WHOLESOMENESS OF FOOD IRRADIATED WHITH DOSES ABOVE 10kGy. Switzerland: World Health Organization (1997). INAC 2007, Santos, SP, Brazil.
