Protocolo
BACTERIOPLANKTON CHEMICAL COMPOSITION ANALYSIS
BACTERIOPLANKTON CHEMICAL COMPOSITION ANALYSIS
PROTOCOL
Materials
- Whatman Type GF-F filter, 0.7 µm, 47 mm dia (3 filters per sample)
- Whatman Type GF-A filter, 1.6 µm, 47 mm dia (3 filters per sample)
- Any other larger pore size filter in case of high suspended solids concentration
- 5 L polypropylene amber container (Nalgene). Acid washed
- 20% Paraformaldehyde (PFA) or P+G or GLY-TE (cells fixation for cytometry or epifluorescence)
- Glass Filtration unit (Millipore, 47 mm dia). Acid washed or oven treated
- Forceps (acid washed)
- Aluminum foil pocket
Sampling
- Rinse the polypropylene amber acid washed container with water from the aquatic system
- Collect 5L superficial sample in the polypropylene rinsed container
- Refrigerate the sample until filtration (no longer than 12h)
- Filtration: see figure below (manipulate the filter always with clean forceps)
- Prepare the filters:
- burn the GF-F and GF-A filters during 4 h at 550°C.
- Weight the GF-F and GF-A filters and record the filter weight
- Preserve a sub-sample of the unfiltered water for bacterial abundance analysis (see below filtration product analyses)
- Pre-filter sample through Whatman Type GF-A filter (1.6 µm) and preserve a sub-sample of the “filtration product I” larger for bacterial abundance analysis (see below filtration product analyses)
- Keep the GF-A filter (see below the procedure to dry it).
- Filter the product of filtration from “step c” through Whatman Type GF-F filter (0.7µm). Preserve a sub-sample of the filtration product for bacterial abundance analysis (see below filtration product analyses)
- Keep the GF-F filter (see below the procedure to dry it).
- Collect the product of filtration from “step e” in acid washed 50 ml vials for further analyses (see below)
- Repeat the steps a to g 2 times (one filter per C, N and P analysis)
Filter storing
- Oven dry (approx. 60°C) the filters GF-F and GF-A that contains the sample, until constant weight.
- Cool the filter in the desiccator
- Record the filter weight
- Fold each filter with the bacteria facing inside and place them in individual aluminum foil pocket.
- Write the sample ID on the edge with a pencil
- Keep at -20ºC until analysis
Filtration product analysis
- Bacterial abundance: determine bacterial abundance from the sub-samples filtration products by flow cytometry or epifluorescence microscopy. Record bacterial abundance.
- C, N, P (filters and < 0.7mm water) analysis: Samples will be analyzed at André Amado Lab.
- Fill the Tabla para Muestreo de la composición química
- Mail samples to:
Laboratorio de Ecologia Aquatica, Instituto de Biociencias (ICB)
Universidade Federal de Juiz de Fora (UFJF).
Rua Jose Lourenço Kelmer, s/n – Campus Universitario, Bairro Sao Pedro
CEP (ZipCode): 36036-900
Juiz de Fora, Minas Gerais, Brazil.
Figure 1.