Introduction. Blastocystis hominis is a common polymorphic intestinal protozoan in humans. Pathogenic and non-pathogenic subtype existence is still under investigation. Materials and methods. A simplified medium consisted of 2.5 ml agar slant and Stone’s modification of Locke’s solution supplemented with 20% peptone and 30% heat-inactivated horse serum, giving a complete medium volume of 6.5 ml, was newly developed. A PCR-based assay used single strand conformation polymorphism (SSCP)/sequencing to detect Blastocystis both directly from human faeces and in parasitic cultures. Mixed infections with Blastocystis and other enteric parasites were microscopically excluded. Xenic cultures (with rice starch) were used to confirm single infections and examine Blastocystis morphology. Using cultivation and PCR-SSCP, parasite’s genotype/phenotype association was explored among immunocompetent individuals with Blastocystis alone in regards to possible clinical symptoms. Results. The new medium was used to cultivate 25 positive and 20 negative for Blastocystis samples. In comparison to Robinson’s medium, an established biphasic medium, it proved to be equally capable of isolating Blastocystis and checking amoeboid presence. Its potential use in Blastocystis maintenance was speculated. The PCR-SSCP method was applied to 45 positive and 30 negative for Blastocystis faecal samples and proved to be highly sensitive and specific. This protocol could classify Blastocystis among the nine established subtypes. Of 1,913 faecal specimens, Blastocystis infection rate was microscopically estimated at 3.5% in a Greek population. Thirty-two strains, isolated from asymptomatic single-infected individuals, presented only vacuolar (and granular) forms during culture. In 50% of these, subtypes 1, 2, and 7 were found; in the remaining 50%, subtype 3 was identified. Subtype 3 was also detected in 12 mono-infected patients with minor chronic gastrointestinal symptoms. One patient with acute urticarial lesions together with minor gastrointestinal symptoms was infested with Blastocystis sp. subtype 3 additionally producing amoeboid forms throughout cultivation. Likewise amoeboid Blastocystis appeared in cultures of 11/12 symptomatic subtype-3 isolates. In symptomatic isolates, amoeboid forms were discriminated from other forms and irregular Blastocystis, with plasmotomy having possibly been seen. A subtype-4 strain presented as amoeboid was isolated from a symptomatic patient. Conclusion. Simple PCR-SSCP method might contribute towards studies on Blastocystis molecular epidemiology and clinical importance. An amoeboid symptomatic subtype-4 isolate was firstly identified. There was a difference in morphology between subtype-3 asymptomatic and symptomatic isolates. Thus, potential pathogenic role of the preponderant human subtype 3 (or clone) presenting as amoeboid in cultivation could be suggested. Currently, amoeboid presence checking in short-term cultures using biphasic medium such as the user-friendly alternative simplified medium might provide a simple diagnostic tool to screen patients suffering from clinical symptoms related to parasite for potential pathogenic Blastocystis subtype or clone.