Tetrahymena pyriformis reproduction inhibition test to soil


Tetrahymena pyriformis microscopic animal is a sensitive test organism, its reproduction is inhibited in the presence of toxic substances. Soil toxicity can be monitored by the change of the number of animals (cell number).

The test method was developed by Gruiz and Leitgib (2006) and later on the method was further developed by BME ABÉT’s Environmental Microbiology and Biotechnology Group.

The methodology:

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

Inoculum: 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:

1.5 g soil sample is introduced into 100 ml-es Erlenmeyer glass and sterilised (in the autoclave at 121 °C for 20 minutes). 30 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. Two-three replicates are applied. Uncontaminated soil is used as control or in case it is not available then forest soil is used instead. The glasses are shaken at 100 rpm, at room temperature (20-25 °C), in the darkness.


The reproduction curve of the single cell 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 carefully mixed. The cell number is determined by microscopy: the number of cells in 2 μl 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.


Gruiz K., Leitgib L.: Tetrahymena pyriformis reproduction inhibition test for ecotoxicity testing of contaminated soil. LOKKOCK project study, BME MGKT, Budapest, 2006. (In Hungarian)

Feigl, V. Tetrahymena pyriformis reproduction inhibition test to soil (in Hungarian) https://www.enfo.hu/keptar/5270