Tetrahymena pyriformis reproduction inhibition test to water samples (surface- and groundwater, leachates, waste waters)

Tetrahymena pyriformis is a freshwater, eucariotic, unicellular protozoa belonging to the ciliates. The body of Tetrahymena pyriformis is generally 50-60 µm long and 30 µm wide and is pear-shaped, a characteristic from which the name of the species is derived. Various modifications of the form are possible in old or stressed cultures. Eighteen longitudinal rows of cilia calles kinetics cover the cell surface. It is the most common protozoan model used in toxicological studies. it is characterised by short life cycle, which allows easy cultivation in suitable laboratory conditions. The toxic effects of substances on several generations can also be tested.

Monitoring the change of the number of animals (cells) the toxic effect of chemical substances and environmental elements (soil, sediment, surface and underground water, waste water, etc) may be measured. The test method was developed by Gruiz and Leitgib (2006) and later on further developed by BME ABÉT’s Environmental Microbiology and Biotechnology Group.

Performace of the test

Nutrient solution (PP) preparation: 10g tripton, 1g yeast extract, 1000 cm3 tap water, sterilised in autoclave at 121 °C for 20 minutes.

Inoculum preparation: The test animal is kept by weekly inoculation. 5 ml sterile PP nutrient solution is inoculated with 100 μl cell suspension then it is thermostated at 20 °C, in the darkness.

Test procedure:

15-15 ml each, water sample or dilution series sample is introduced into a 100 ml sterile Erlenmeyer glass. 15 ml PP nutrient solution and 156 μl antibiotic solution (0.2% Penicillin, 2% Streptomycin, 1% Nystatin) is added into each glass, then it is inoculated with 600 μl 6 days old homogenous Tetrahymena culture. Three replicates are applied. Dilution series are prepared from the chemical substance. Sterile tap water is used as control. The glasses are shaken at 100 rpm, at room temperature (20-25 °C), in the darkness.

Evaluation of the results:

The reproduction curve of the unicellular animal could be plotted based on 4 measurements during 72 hours. For cell counting the cell suspension is homogenised (carefully mixed), then 500 μl is extracted from it and 20 μl 1% formalin solution is added into the 500 μl suspension and it is again carefully mixed. The cell number is determined by microscopy: the number of cells in 2 μl cell suspension pipetted onto the Bürker chamber is counted. If necessary, the suspension is diluted with water. The slope of the curve plotted on the exponential section of the reproduction curve is compared with the uncontaminated control.