The usefulness of changing focus during examination using Gram staining as initial diagnostic clue for infective tuberculosis

Gram staining is a useful technique for detecting bacteria but is highly questionable in detecting Mycobacterium tuberculosis. Its detection generally requires special staining, such as Ziehl–Neelsen staining. We experienced three cases in which tuberculosis was first suggested by Gram staining of sputum or pus, confirmed by Ziehl–Neelsen staining, and diagnosed by polymerase chain reaction or culture. To find colorless tubercle bacilli in clinical samples with various organisms, varying the focus to slightly longer and shorter during study of the slides is indispensable. We present criteria for detecting infective pulmonary tuberculosis in Gram staining. First, in the ordinary focus, weakly stained, thin, gram-positive bacilli are found; second, with a slightly longer focus distance, the thin, cord-like, conspicuous gram-positive bacilli can be observed; and third, with a shorter focus distance, the gram-positive bacilli have changed into the brightened, colorless, or ghost ones. Four laboratory technologists each evaluated 20 Gram-stained samples after being lectured on the criteria, with no prior information about the sample. They accurately evaluated the presence of the bacilli in Gram-stained preparations in more than 90% of samples containing 3+ bacilli on Ziehl–Neelsen staining. Gram staining is available as an easy and rapid initial clue to recognize highly infective tuberculosis.