Blastocystis hominis provides major challenges for laboratory diagnosis due to its polymorphic nature in wet mounts which can result in confusion with other protozoa, yeast or even fat globules. Studies revealed that simple smears were less sensitive than in vitro cultivation using different media for the detection of B. hominis in stool specimens. Cultures of B. hominis are usually enriched by different types of sera to enhance growth and multiplication of the parasite.The aim of this study is to assess the use of two sera types other than horse serum that is commonly used in culture media for the growth, multiplication and detection of B. hominis in examined stool samples and comparing the results with those obtained using horse serum. Fifty stool samples were collected from patients suffering from different colonic manifestations attending Cairo University Hospitals. The samples were freshly cultured in three different culture media using horse serum (in Jones' medium), donkey serum (as a modification in Jones' medium) and human plasma (in modified Pavlova's medium) in adequate preparations. Cultures were then left for incubation and examined by direct microscopy to detect Blastocystis hominis. The results showed of 50 stool samples studied, 18 samples (36%) were positive results for B. hominis. The number of positive results obtained by horse serum, donkey serum and human plasma were 13,18and 11 respectively. Paired comparisons were made between each 2 cultures with each culture set as a reference once to detect the most appropriate one for diagnosis. When horse was set as the reference method, donkey serum showed a sensitivity of 100% and specificity of 86.5% with a 90% agreement between the 2 methods. While human plasma showed a sensitivity of 46.2% and specificity of 86.5% with an agreement of 76%. In addition, the vacuolar form was the commonest pattern observed in this study throughout all the three cultures.