Tuberculosis is a potentially deadly infectious disease caused by the Mycobacterium tuberculosis (M. tuberculosis). Tuberculosis is diagnosed by proving the M. tuberculosis in sputum samples based on the results of acid-resistant staining, culture, and nucleic acid amplification tests. However, there is a report that the detection rate of M. tuberculosis is low in acid-resistant staining using tissue specimens. It has been suspected that the cause is a potential loss of acid resistance by the organic solvents used for tissue specimen preparation. Therefore, this study was pursued to find out if Gram staining and fluorescent staining in addition to acid-resistant staining would be helpful in diagnosing tuberculosis. We used four tissue (lung, small intestine, large intestine, and lymph node) samples with chronic granulomatous inflammation observed in HE staining and positive results in real-time PCR. These detection rates and staining properties were investigated through microscopic examination using the Ziehl-Neelsen, Gram, and Auramin rhodamine staining. In this studies, M. tuberculosis were observed by Ziehl-Neelsen, Gram, and Auramin rhodamine staining in all four samples. In the evaluation of clinical microbiology proficiency testing (CMPT), the Ziehl-Neelsen and Gram staining were the same result, but the Auramin rhodamine staining was relatively low. These data indicated that Gram staining is useful for detecting M. tuberculosis in formalin-fixed tissue specimens. Therefore, if the Ziehl-Neelsen and Gram staining are combined as the M. tuberculosis staining method in tissue specimens, a better direction may be provided for tuberculosis diagnosis.