One liter of water is collected with a
plastic bucket for surface samples and with a Niskin or Van Dorn water sampler
for subsurface samples. The water sample is fixed with
glutaraldehyde at a final concentration of 1%. Fixed samples are stored in a cool, dark
place, shielded from light. It is recommended to store in a refrigerator.
After the fixed samples are weighed, they
are placed on a stone table (Figs. 1 and 2) or other non-shaking surface for at
least 24 hours to allow the phytoplankton to settle (Fig. 3).
Then, the supernatant phytoplankton-free
seawater is removed using a siphon (Fig. 4).
In the Plankton Laboratory, after concentrating
in a 1 L container, it is placed in a 110 mL bottle, and precipitation again. After
that, the supernatant is removed using a siphon. Samples are concentrated until
the final concentration is in a 20 mL bottle (Fig. 5).
The weight of the concentrated sample is
The value that subtracted respectively the
weight of the bottle used in the first field and the weight of the bottle used at
final is used to determine how many times the sample was concentrated and the
concentration factor of the sample.
Preparation for microscopic examination of samples
The concentrated sample is also stored in
the refrigerator to shield from light. At the time of samples microscopic
examination, the sample is gently stirred and a fixed volume (about 2 mL) is
collected with a pipette. It is placed on a glass slide for phytoplankton
counting, which has a mesh pattern on the bottom (Fig. 6).
The glass slides for phytoplankton counting
have a frame that can hold a 2 mL sample, into which the concentrated sample is
A cover glass that fits this large frame is
also commercially available.
When the sample is inserted between the
glass slide frame and the cover glass, and the cover glass begins to float,
capillary action causes the cover glass to rotate so that it fits over the
glass slide frame (Figs. 7 and 8).
Observation under the inverted microscope
Allow the phytoplankton to stand in the
glass slides until it precipitates (about 10 minutes), then observe it under an
inverted microscope at about 200-400x (Figs. 9 and 10).
During counting, the value of how many
cells are present when counting the mesh is taken, and the number of observed
cells per overall basal area is used to calculate how many cells were contained
in the entire concentrated sample collected (Fig. 11).
For phytoplankton, identification is made
to the species level based on Hasle and Syvertsen (1997) and Horner (2002). For
the dinoflagellate Karenia selliformis, the number of chloroplasts in
the cell is useful for species identification (Iwataki et al. 2021). For
ciliates, the counts are divided into two groups: tintinnids and oligotriches.
Nanoplankton and siliceous flagellates are also counted. A minimum of 300 cells
per sample will be counted. After counting, the cell density (cells mL-1)
of each taxon in the sample is calculated.
Hasle, G.R. and Syvertsen, E.E., 1997. Marine
diatoms. In: Tomas, C.R. (Ed.), Identifying marine phytoplankton.
Academic Press, San Diego, pp. 5–385.
Horner, R. A. (2002) A Taxonomic Guide To
Some Common Marine Phytoplankton, Biopress Limited, United Kingdom
Iwataki, M., W. M. Lum, K. Kuwata, K.
Takahashi, D. Arima, T. Kuribayashi, Y. Kosaka, N. Hasegawa, T. Watanabe, T.
Shikata, T. Isada, T. Y. Orlova and S. Sakamoto (2021) Morphological variation
and phylogeny of Karenia selliformis (Gymnodiniales, Dinophyceae) in an
intensive cold-water algal bloom in eastern Hokkaido, Japan in
September–November 2021. bioRxiv preprint. https://doi.org/10.1101/2021.12.13.472515