Bacillus is a predominant genus of bacteria isolated from tobacco. The Gram stain is the most commonly used and most important of all diagnostic staining techniques in microbiology. In order to help confirm the Gram positivity of Bacillus isolates from tobacco, three methods using the chemical differences of the cell wall and membrane of Gram-positive and Gram-negative bacteria were investigated: the KOH (potassium hydroxide), the LANA (L-alanine-4-nitroanilide), and the vancomycin susceptibility tests. When colonies of Gram-negative bacteria are treated with 3% KOH solution, a slimy suspension is produced, probably due to destruction of the cell wall and liberation of deoxyribonucleic acid (DNA). Gram-positive cell walls resist KOH treatment. The LANA test reveals the presence of a cell wall aminopeptidase that hydrolyzes the L-alanine-4-nitroanilide in Gram-negative bacteria. This enzyme is absent in Gram-positive bacteria. Vancomycin is a glycopeptide antibiotic inhibiting the cell wall peptido-glycan synthesis of Gram-positive microorganisms. Absence of lysis with KOH, absence of hydrolysis of LANA, and susceptibility to vancomycin were used with the Gram reaction to confirm the Gram positivity of various Bacillus species isolated from tobacco. B. laevolacticus excepted, all Bacillus species tested showed negative reactions to KOH and LANA tests, and all species were susceptible to vancomycin (5 and 30 µg).
Flue-curing is a post harvest conditioning process which strongly affects the tobacco leaf chemistry, and consequently the chemical properties of tobacco smoke. Several studies identified the major changes in tobacco chemistry occurring during flue-curing. It is not known how flue-curing contributes to changes in bioactivity of cigarette smoke condensate (CSC). In this study, tobacco leaves collected throughout the twelve days of flue-curing were used to prepare cigarettes that were smoked to generate CSC samples. The assessment of mutagenicity was performed using the Bacterial Reverse Mutation / Ames test with Salmonella typhimurium TA98 in the presence of S9 metabolic activation. CSC from cured leaves were significantly more mutagenic than CSC from uncured leaves. The number of revertants was positively influenced by the duration of the curing. The effect of the duration of curing on the number of revertants was more pronounced with increasing CSC concentration.