Optimization of preservation and processing of sea anemones for microbial community analysis using molecular tools.
For several years, knowledge on the microbiome associated with marine invertebrates was impaired by the challenges associated with the characterization of bacterial communities. With the advent of culture independent molecular tools it is possible to gain new insights on the diversity and richness of microorganisms associated with marine invertebrates. In the present study, we evaluated if different preservation and processing methodologies (prior to DNA extraction) can affect the bacterial diversity retrieved from snakelocks anemone Anemonia viridis. Denaturing gradient gel electrophoresis (DGGE) community fingerprints were used as proxy to determine the bacterial diversity retrieved (H'). Statistical analyses indicated that preservation significantly affects H'. The best approach to preserve and process A. viridis biomass for bacterial community fingerprint analysis was flash freezing in liquid nitrogen (preservation) followed by the use of a mechanical homogenizer (process), as it consistently yielded higher H'. Alternatively, biomass samples can be processed fresh followed by cell lyses using a mechanical homogenizer or mortar &pestle. The suitability of employing these two alternative procedures was further reinforced by the quantification of the 16S rRNA gene; no significant differences were recorded when comparing these two approaches and the use of liquid nitrogen followed by processing with a mechanical homogenizer.
1] Departamento de Biologia &CESAM, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal [2] Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira n.° 228, 4050-313 Porto, Portugal.
Joana
J
Rocha
Departamento de Biologia &CESAM, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal.
Francisco J R C
FJ
Coelho
REQUIMTE, Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n. ° 228, 4050-313 Porto, Portugal.
Luísa
L
Peixe
Departamento de Biologia &CESAM, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal.
Newton C M
NC
Gomes
Departamento de Biologia &CESAM, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal.
Ricardo
R
Calado
D000818:Animals; D015373:Bacterial Typing Techniques; D015925:Cryopreservation / Q000379:methods*; D016172:DNA Fingerprinting; D058645:Denaturing Gradient Gel Electrophoresis; D005798:Genes, Bacterial; D064307:Microbiota / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012615:Sea Anemones / Q000382:microbiology*; D013048:Specimen Handling / Q000295:instrumentation / Q000379:methods
D012336:RNA, Ribosomal, 16S