In order to study specific metabolic pathways in microorganisms is crucial to have complete control on the components of the growth medium. This protocol describes the components and mode of preparation of a minimum medium (low nutrients medium) of defined composition, suited for marine holozoans (named "modified L1 medium" - mL1). mL1 medium can be used for cultivation of different marine holozoans (i.e. Ichthyosporea and Corallochytrea), allowing to completely control the components (for example: carbon source, nitrogen source, etc). mL1 medium is based on the L1 medium recipe publically available at the National Center for Marine Algae and Microbiota (http:\/\/ncma.bigelow.org\/algal-recipes).