How to verify axenicity - a method comparison

An axenic culture is defined as a culture containing no other living organism except the strain of interest. In some phycological research areas, such as biochemistry and physiology, axenic growth is essential to avoid misinterpretations caused by contaminants. If axenicity is required, reliable assessment tools are obligatory to verify the culture's axenic state. We compare the available methods to assess axenicity. We first purified Limnospira fusiformis cultures with an established series of treatments. The presumable axenic cultures were then tested for their axenic state by applying conventional tests on LB-agar-plates, 16S rRNA gene amplicon sequencing, flow-cytometry and epifluorescence microscopy. Only the plate tests indicated axenic conditions. 16S rRNA gene amplicon sequencing demonstrated its superiority by not only being an efficient tool for axenicity testing, but also for identification of persistent contaminants. Although classic plate tests are still commonly used to verify axenicity, we found the LB-agar-plate technique to be inappropriate. Cultivation-independent methods are highly recommended to test for axenic conditions. Sequencing was performed at the Joint Microbiome Facility of the Medical University of Vienna and the University of Vienna under the project ID JMF-2011-B.