For cellular analysis, the buccal mucosa was brushed 10 times using a sterile cytobrush (Histobrush, Hardwood Products Company, Guilfor, ME, USA) to obtain cells from buccal vestibule in SD and NSD subjects. Each dipping site of Naswar was brushed to compare the obtained cells with the control subjects. The obtained specimen was spread on the prepared glass and fixed with 80% methanol after drying for 20 min; 3 slides were prepared per patient. Subsequently, Giemsa staining was performed with a 10% solution for 20 min. After washing and drying, the DPX mounting medium (Merck KGaA, Darmstadt, Germany) was poured and pressed on the slide, followed by storage at room temperature for microscopic analysis (Binocular NSL CX23, Olympus, Japan). The cytological smears were observed microscopically with a consultant oral histopathologist (A.M.). The slides were viewed under oil immersion lens at 1000x magnification to identify and then count the micronuclei per cell. Careful counting existed to avoid overlapping of fields and repeated counting of same cells. A number of 1000 cells were counted for each case and Tolbert et al. criterion was followed for identification and scoring of micronuclei [20].

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