A Simple Method For Plating and Cloning Ciliates and Other Protozoa

Abstract

A simple method is described for plating and cloning ciliates and other protozoa, based on a principle differing from that traditionally used for plating and cloning bacteria and other microorganisms. This procedure, referred to as te silicone-oil-plating-procedure (SOPP), involves vortexing small volumes of culture medium containing protozoa with larger volumes of a non-toxic silicone oil and plating the resulting unstable emulsion in small plastic petri plates. Discrete microdroplets of culture medium form containing protozoa entrapped and immobilized between the hydrophobic surfaces of teh plastic petri dish and the oil. Protozoa, isolated by this method grow, divide, and multiply to form clones. The procedure may be used for plating and cloning protozoa in bacterized and axenic culture. Variations of te basic method may be applied to isolating protozoa from the wild, washing protozoa to remove microorganisms, screening for potential mutants, and for replica plating.

The feeding behavior of Spumella sp. as a function of particle size: Implications for bacterial size in pelagic systems

“…After two days flagellates were isolated using the silicon-oilplating procedure described by Soldo & Brickson (1980). A 0.5 ml sample of the enrichment with additional bacteria was added to 3 ml of silicon oil contained in a 10 ml glass vial .…”

Effects of prey abundance and light intensity on the mixotrophic chrysophyte Poterioochromonas malhamensis from a mesotrophic lake

“…Water samples were collected using a 10-mm mesh plankton net and transferred to the laboratory in sterile plastic bottles and enriched with plate-cultured bacteria. The flagellate was isolated using the plating technique described by Soldo & Brickson (1980) modified by replacing silicone oil with mineral oil. Identification as the mixotrophic chrysophyte P. malhamensis was based on its size (6±8 mm diameter), one long and one short flagellum, possession of a chloroplast in addition to digestive vacuoles in the cytoplasm, and the presence of a cup-shaped lorica (Patterson, 1996).…”

The Invasive Nature of an Infectious Bacterial Symbiont

“…Washed, xenosome-free P. acutum 110-3 (25,00O/ml) were mixed with a freshly prepared suspension of xenosomes at a concentration of 10,000 xenosomes per protozoan in instant sea water (ISW) (d = 1.01 5 g/cc) at room temperature. At intervals samples were removed, diluted 100fold in fresh culture medium [7] to non-infective levels and plated by the silicone oil plating procedure (SOPP) [9]. For each time period, 20 clones were isolated, subcultured in fresh culture To whom correspondence should be addressed.…”

“…This procedure was similar to that described above, except that xenosome-free P. acuturn 263-20 was infected with freshly prepared suspensions of killer (1 10-3xp) and non-killer (263-2Oxp) xenosomes mixed together in various proportions at a final concentration of 10,000 xenosomes per protozoan in ISW. After 2 h preincubation at room temperature, the suspensions were diluted 100-fold with fresh culture medium and plated by the SOPP [9]. For each mixture, 10 to 20 clones were isolated and subcultured.…”