There is the potential for smokers of ventilated cigarettes to block the ventilation holes either accidentally or deliberately thereby altering the smoke deliveries from those obtained by standardized machine smoking. One way in which the holes can be blocked is by inserting the cigarette into the mouth so that the holes are partially or completely blocked by the lips of the smoker. We have assessed to what extent this occurs amongst Canadian smokers by measuring the saliva patterns on 2756 cigarette butts collected in Montreal, Toronto and Vancouver. The butts were a cross-section of brands smoked in Canada. Saliva stains were visualized by treating the tipping paper with ninhydrin solution. The insertion depth was assumed to be the maximum extent of the saliva stain from the mouth end of the tipping. The brand of each cigarette butt was identified where possible as well as whether the filter was ventilated and if so, the distance of the vent holes from the mouth end. The butt lengths were also determined. Of the 2756 butts collected, 2232 had lip imprint patterns that could be visualized with ninhydrin solution. 56.2 % of the butts with measurable insertion depths, and which could be identified by brand, were ventilated. There was no significant difference between the average insertion depths for ventilated and non-ventilated brands (10.6 AA± 3.6 and 11.0 AA± 3.6 mm respectively). It was estimated that for the ventilated brands between 3.7-10.3 % of the butts could have had the vents blocked completely for at least one puff, 13.8-20.4 % of the butts had vents that could have been partially blocked and 75.9 % of the butts showed no sign of any vent blockage during smoking.
Canadian tobacco was flue-cured using two different heating systems: direct-fired in which the exhaust gases were in contact with the tobacco and indirect in which only hot air, via a heat exchanger, contacted the tobacco. The concentrations of tobacco-specific nitrosamines (TSNAs) in tobacco cured by indirect heating did not increase during curing and were in the range 0.25-0.35 ppm. There were no changes in TSNA concentrations (range 0.13-0.3 ppm) in tobacco cured by direct firing during the first six days (0-144 h) of curing. However between 168 and 264 h, significant increases in TSNAs occurred (up to 1.91 ppm). TSNA concentrations in leaves at the bottom of the plant were significantly higher than in those found at higher plant position. There were no significant differences in TSNA concentrations in tobacco cured on different farms. The TSNA concentrations in tobacco cured by indirect heat were 87% ± 5% lower than in tobacco cured by direct heat. Subsequent processing of tobacco did not change the relative concentrations of TSNAs.
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).
Protein transfer in tobacco smoke has been studied using the protease, Savinase™, as a model protein. Mainstream and sidestream smoke were collected from cigarettes to which Savinase had been added at various concentrations. Savinase was extracted from the smoke condensate with an organic solvent system before being precipitated and further identified by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting. The detection limit of the method, based on addition of Savinase to the smoke condensate, was 25 µg in mainstream and 100 µg in sidestream smoke. At a Savinase concentration of 6000 µg per gram of tobacco, the methodology allows the detection of protein transfer as low as 0.009% and 0.054% in mainstream and sidestream smoke, respectively. Using this approach, it was shown that there is no detectable Savinase in the mainstream and sidestream smoke of filtered and unfiltered cigarettes containing up to 6000 µg of Savinase per gram of tobacco. These facts strongly suggest that there is no significant transfer of protein from tobacco into cigarette smoke.