Material and methods



1. Meteorological conditions during the period of observations
  • Valamo Island is characterised with the average annual temperature 3.6 oC. Average air temperature in February is -8.6oC, in July 16.7oC. Average annual number of days with positive temperature - 165 (Gagarin et al., 1998: 1998. Proc. St. Petersburg nat. Soc., s.1, v. 92, p. 4-16).

    2. Sampling sites
    Fig.1. Selected sampling sites.
  • During the study the lake was covered with a net of 250 single samples. 5 sites, representing rather contrast ecotops, were selected for monitoring study. At these sites samples were collected periodically, in different years and seasons. Reduced map of sampling is shown at the Fig.1.
  • Samples of the upper layer of bottom sediments were colected with glass or plastic cans. 5-10 mm of the sediments with near-the-bottom water were collected. Cores of sediments were collected either with glass tubes (for best visual study of the sediments structure) or with plastic tubes, 60 mm in diameter, perforated with a row of openings at one side. Subsamples from the cores were collected with the glass tubes via these openings.


  • Sampler. This one is done of black plastic. Subsamples from the core are collected with the glass tubes into sterile 40 mm Petri dishes.

    3. Study of amoebae fauna

  • Samples, collected for faunistical study were inoculated to the set of media, described by Page (1988: A new key to freshwater and soil gymnamoebia. FBA.). Most favourable were found to be NNE and CPA (op. cit.), All samples were inoculated to the 40 mm Petri dishes with and without overlay of PJ or soil extract (op.cit.). To recover large species, samples were inoculated to 100 mm Petri dishes with PJ solution and 2-3 weat grains.
  • Light-microscopical investigation was carried out using the "MBI-15-2" photomicroscope equipped with phase contrast optics and UB-1 microscope (PZO) equiped with Nomarski' contrast. Permanent preparations were fixed with Bouin's solution, stained with iron haemotoxylin and embedded into the Canadian balsam.
  • For electron microscopy amoebae were treated under the room temperature with one or all of listed procedures: (1) fixed in 4% glutaraldehyde (20-40 min), washed in buffer (3*5 min), postfixed in 1% osmium tetroxide (60 min); (2) 0.5 % osmium tetroxide (15 min) which was replaced with 1% osmium tetroxide (60 min); (3) 0.5% osmium tetroxide (5 min), washed in buffer (5 min), 4% glutaraldehyde (20 min), washed in buffer (3*5 min); postfixed with 1% osmium tetroxide (60 min). All the fixatives and washing were made using the phosphate buffer (pH 7.4).
  • After dehydration in ethanol series amoebae were embedded in the Epon-Araldite resin. Sections were stained with saturated solution of uranyl acetate in 50% ethanol and with Reynolds' lead citrate and examined in a "Tesla BS-500" and "Hitachi H-300" electron microscope.

    4. Differential counting of amoebae
    An original counting method, illustrated below was elaborated and applied in course of this work since 1992, however actually was published only in 1998 (delay of the journal appearance) (Smirnov et al., 1998. Proc. St. Petersburg nat. Soc. s.1, v. 92: 74-81. ). This method was found to be much more suitable for field work than routine MPN methods. Verification of this method, done by mean of repetitive countings from the same well-mixed, homogenous sample indicated the deviation 7-18%, which is much lower that the deviation on the order of magnitude, shown by MPN methods (method by Singh (1946: Ann. Appl. Biol. 33: 112-119) was used). Very similar approach was suggested by Anderson & Rogerson (1995: Europ. J.Protistol. 31: 223-233)

    Method of amoebae counting, used in this work (above ref: Smirnov, 1998)