Protocol to Establish Axenic Cultures for Diatoms of Fresh Water

Diatom axenic cultures have great potential importance in ecological, whole genome and other single genera based studies. The generation of axenic diatom cultures like other cell culture requires special treatment and requirement of macro, micronutrients vitamins and pH. However, since silica is the most important component of diatom cell wall it is most important component of the media too. The f/2 media with little modification served as the best media for fresh water diatoms collected from water bodies of Northern western India with culture conditions standardized at temperature 22C, light intensity 18000 lumen per m2, 16 hours/8 hours of lightdark conditions. The present study demonstrates the protocol to hinder the growth of fungi, bacteria and other green algae which contaminates the diatom culture. It was observed that antibiotic mixture of penicillin G and streptomycin in the ratio of 10.5 removed most of the bacterial growth in the culture media. The fungal spores were very rigid to remove from the culture plate and treatment with amphotericin B (2.5mg/L) did not reduce the appearance of fungal mycelium, however multiple centrifugation and washing with milliQ water gave relief from fungal spores. The axenicity of diatom culture is further confirmed using 16S rRNA gene based primers which showed that on blasting the amplicon the multiple sequence alignment had phylogeny resembling different diatom genera and species.