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It is challenging to determine whether Bacillus species other than Bacillus anthracis cause infections. Pseudo and true outbreaks of Bacillus spp. have been noted. Here, we present a molecular analysis of a Bacillus spp. pseudo-outbreak caused by contaminated culture tubes containing Stuart medium.Between January and March 2015, a high percentage of Bacillus spp. was isolated from the wound samples of inpatients at the Karabuk University Hospital, and an outbreak was suspected. Environmental and staff nasal samples were cultured aerobically, and Bacillus spp. were isolated from some of them. However, the isolation of Bacillus spp. in throat cultures of outpatients suggested contamination caused by culture tubes containing Stuart medium. We examined two lots of culture tubes used in the hospital. Although the culture tubes' expiry date and storage conditions were suitable, Bacillus spp. grew in one of these lots. A total of 47 Bacillus spp. isolated during this period were identified, and the clonal relationship among the isolates was investigated by arbitrarily primed polymerase chain reaction.Twenty-seven strains were identified as Bacillus megaterium and 20 as Bacillus firmus. Of the four strains isolated from the Stuart medium, two were identified as B. firmus and the other two were B. megaterium. Two B. firmus strains isolated from the Stuart medium and two B. firmus strains obtained from the coronary intensive care environmental samples were matched and clustered within the same genotype. We recalled all culture tubes containing Stuart medium. After another brand's culture tubes were distributed, no growth was observed. It was then understood that the pseudo-outbreak source was contaminated culture tubes containing Stuart medium.Microbiological controls of medical materials and equipment should be regularly checked to prevent outbreaks (true or pseudo).