Sputum smear microscopy is an easy, inexpensive, and rapid method for detecting tubercle bacilli when there are more than 10,000 bacilli/ml in the original sputum. Furthermore, because the microscopic method provides not only quantitative, but also qualitative information, such as the shape of bacilli, it has remained significant. We have previously developed and reported panel test slides made from polyacrylamide-based artificial sputum (PBAS) mixed with both cultured THP-1 cells and nonpathogenic mycobacteria. In this paper, we report an improved preparation method for PBAS for panel test slides that provides a simplified method and enhanced availability with high consistency in each grade and in which only negative PBAS is prepared from polyacrylamide and cultured THP-1 cells and mixed with graded formalin-fixed Mycobacterium tuberculosis solution (FFTBS) containing oral flora and Pseudomonas aeruginosa on the slides. In the smears prepared using this improved method, the numbers (average ± standard deviation [SD]) of acid-fast bacilli (AFB) in 300 fields (2-by 3-cm smear) in eight smears of each grade ranged from 5 to 9 (6.4 ± 1.4), from 59 to 88 (74.6 ± 10.0), from 503 to 912 (705.0 ± 145.7), and from 1,819 to 3,256 (2133.3 ± 478.0) in ±, +, ++, and +++ smears, respectively. In addition, this preparation method provided high similarity to the microscopic appearance of bacilli and background seen in the actual patient sputum, with high feasibility. These results revealed that our new PBAS had high authenticity in the appearance and consistency in each grade, which could make it valuable as a reliable artificial sputum for the training of microscopists.