Bacillus subtilis growth inhibition test to soil with agar diffusion method

Test type: One species, acute, bacterial ecotoxicity assay

The test was developed by BME’s Environmental Microbiology and Biotechnology group for the assessment of metal contamination. The Bacillus subtilis test-organism is moderately sensitive to toxic metals.

 The principle of the method: Agar plates are prepared from the environmental sample (contaminated soil) then placed on the surface of the nutrient agar disc containing the test organism. Generally, the contaminant diffuses into the agar plate and function of its amount and toxicity it inhibits germination and growth of the uniformly distributed Bacillus subtilis. The diameters of inhibition growth zones are measured to determine the inhibition.

Description of the agar diffusion method: Bacillus subtilis is cultured and grown in 20 ml meat nutrient solution (inoculum), incubated at 28 ^oC, for 24 hours. The experiment is performed in 30*30 cm glass trays, to which 20 mL inoculum mixed in 100 ml meat agar at 50 ^oC is added.  To prepare uniformly thick nutrient agar one should ensure a fully horizontal surface for the nutrient agar. After solidification of the nutrient agar the sample discs should be placed onto the surface of the nutrient agar.

The sample discs for the sensitivity test are prepared as follows: a mixture of 0.2 ml standard solution and 2 ml melted agar with water is poured in 3.5 cm diameter dishes and after solidification the sample disc is cut in the middle by a screwdriver.

In case of soil samples 1 g soil is mixed into 2 ml agar with water and after hardening we proceed similarly to the standard solutions. After solidification of the agar the sample discs are placed on the surface of the nutrient agar.  The agar plates with the sample discs are incubated at 30 ^oC and the results evaluated after 24 hours.

During evaluation the size and type (inhibition, stimulation) of the concentric area around the sample disc is characterised. The inhibition zone could be separated usually into more rings but always the diameter of the exterior is used for evaluation.

In case of standard solution series the diameter of the inhibition zone is proportional to the contaminant concentration, thus one may conclude from the diameter of the inhibition zone the contaminant amount.

In case of soil samples we are faced with a blend of chemical substances, the behaviour of which in agar is considerably influenced by soil constituents. The solubility and diffusion of various chemical forms of contaminants is different. For this reason the relation between the diameter of the inhibition zone and toxicity is not always quantitative, but it reflects actual toxicity.

For the assessment of contaminated sites pre-screening is recommended with rapid ecotoxicity tests.