Aplicação da técnica de hibridização fluorescente in situ (FISH) para detecção de Bacillus spp

• Autor(a) principal: Santos, Guilherme de Oliveira Ferreira dos
• Data de Publicação: 2011
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/5345
• topic: Oligonucleotídeos; Micro-organismos; Bacillus; FISH; Oligonucleotides; Microorganisms; Bacillus; FISH; CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA::MICROBIOLOGIA APLICADA

Bacteria belonging to the genus Bacillus have physiological plasticity regarding to the conditions of temperature, pH and salinity of the environments in where they are found, such as: water, soil, polluted environments, among others. Under the environmental aspect, these bacteria have the capacity to produce biosurfactants which put them as potential microorganisms for the application of Microbial Enhanced Oil Recovery (MEOR).Technologies economically viable and of growing acceptance, like MEOR, can receive additional informations from the molecular tools about the microbial behavior. In this context, the fluorescence in situ hybridization technique (FISH) is an important tool for detection of microorganisms present in different samples. This technique allows the detection of microorganisms without the need of cultivation. Oligonucleotide probes complementary to the rRNA can be designed with specificities that range from the species level to the domains level. The aims of this work was to apply the FISH technique to detect bacteria of the genus Bacillus and establish the best combination of parameters which combines specificity and good fluorescence signal intensity of the probes utilized. In this way, it was utilized a specific probe for the genus Bacillus (BAC07) and a universal probe for the Domain Bacteria (EUB338). The in silico analysis revealed that the target sequence of probe BAC07 is found predominantly in bacteria of the genus Bacillus, however the possibility of hybridization of probe BAC07 with another member of class Bacilli was not discarded. The parameters analyzed for the application of the FISH technique and evaluation of the specificity of probe BAC07 were: cell fixation method, formamide concentration added to the hybridization buffer and pretreatment of cells with lysozyme. The combination of parameters that ensured the best signal for the probe EUB338 was: cells fixed in paraformaldehyde solution 4%, hybridization buffer containing 35% formamide, without pretreatment with lysozyme; the best conditions for the probe BAC07 were: cells fixed in paraformaldehyde solution 4%, hybridization buffer containing 40% formamide, without pretreatment with lysozyme. A specific detection of bacteria of the genus Bacillus was achieved by probe BAC07. However, the fluorescence signal intensity was very weak when in comparison to the signal of probe EUB338, thereby, for the utilization of probe BAC07 for detection of Bacillus in environmental samples, an enhanced signal is required.