One way to determination of infection frequency in cultured cell that infected with type of Intracellular bacteriaor endogen cell antigenis immunofluorescencestaining, this method is cost and require special equipment. In this study we tried to introduce new staining method for determination of cell frequency in human macrophage that infected with Mycobacterium avium subspecies paratuberculosisand grown in cell culture plate. PMA-differentiated THP-1 cell were infected with mycobacterium at an MOI of 10 (i.e., 10 bacteria per cell), finally after two time of washing cell with PBS,cell was stained based on our protocol. Since heating the cellculture plate for fixing and staining in ziehlnelson staining is not acceptable, this method of staining that was without the use of heating could be a cheap and simple methodfordetermination of cell frequency in human macrophage that infected with Mycobacteriumavium subspecies paratuberculosis.

Keywords