Protocolo

BACTERIOPLANKTON CHEMICAL COMPOSITION ANALYSIS

PROTOCOL

Materials

  1. Whatman Type GF-F filter, 0.7 µm, 47 mm dia (3 filters per sample)
  2. Whatman Type GF-A filter, 1.6 µm, 47 mm dia (3 filters per sample)
  3. Any other larger pore size filter in case of high suspended solids concentration
  4. 5 L polypropylene amber container (Nalgene). Acid washed
  5. 20% Paraformaldehyde (PFA) or P+G or GLY-TE (cells fixation for cytometry or epifluorescence)
  6. Glass Filtration unit (Millipore, 47 mm dia). Acid washed or oven treated
  7. Forceps (acid washed)
  8. Aluminum foil pocket

Sampling

  1. Rinse the polypropylene amber acid washed container with water from the aquatic system
  2. Collect 5L superficial sample in the polypropylene rinsed container
  3. Refrigerate the sample until filtration (no longer than 12h)

  • Filtration: see figure below (manipulate the filter always with clean forceps)

  1. Prepare the filters:
    1. burn the GF-F and GF-A filters during 4 h at 550°C.
    2. Weight the GF-F and GF-A filters and record the filter weight
  2. Preserve a sub-sample of the unfiltered water for bacterial abundance analysis (see below filtration product analyses)
  3. 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)
  4. Keep the GF-A filter (see below the procedure to dry it).
  5. 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)
  6. Keep the GF-F filter (see below the procedure to dry it).
  7. Collect the product of filtration from “step e” in acid washed 50 ml vials for further analyses (see below)
  8. Repeat the steps a to g 2 times (one filter per C, N and P analysis)

Filter storing

  1. Oven dry (approx. 60°C) the filters GF-F and GF-A that contains the sample, until constant weight.
  2. Cool the filter in the desiccator
  3. Record the filter weight
  4. Fold each filter with the bacteria facing inside and place them in individual aluminum foil pocket.
  5. Write the sample ID on the edge with a pencil
  6. Keep at -20ºC until analysis

Filtration product analysis

  1. Bacterial abundance: determine bacterial abundance from the sub-samples filtration products by flow cytometry or epifluorescence microscopy. Record bacterial abundance.
  2. C, N, P (filters and < 0.7mm water) analysis: Samples will be analyzed at André Amado Lab.
  3. Fill the Tabla para Muestreo de la composición química
  4. 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.