Alterations in total bacteria, iodonitrophenyltetrazolium (INT)-positive bacteria, and heterotrophic plate counts of bottled mineral water

Abstract

The quantitative variation in the microflora in bottled mineral waters stored in polyvinyl chloride bottles was studied immediately after bottling and after 7 days storage, over 9 months. The microflora was determined by combining total counts using ethidium bromide, and the number of iodonitrophenyltetrazolium (INT)-positive cells determined with heterotrophic plate counts on R2A for prolonged incubation periods at 22 and 37 °C. Immediately after bottling of mineral water A, the total counts with ethidium bromide varied between 70.0 and 495.0 bacteria/mL. The number of INT-positive bacteria varied between 14.0 and 40.0% counts. The heterotrophic plate counts were higher at 22 °C than at 37 °C and varied between 1.1 and 13.2% and between 0.7 and 9.3% of the total counts, respectively. After 7 days storage, total bacterial counts increased by 1000 times, indicating multiplication of bacteria that were present in low numbers immediately after bottling. The percentage of INT-positive bacteria remained between 2.6 and 39.0% of the total counts but the number of culturable bacteria was higher than the number of INT-positive cells. Similar counts were found in other mineral waters (B, C, and D), but the numbers of INT-positive cells in two of them (B and D) were higher than the heterotrophic plate counts at 22 °C. These results show the presence in the aquifer and (or) bottling system of a flora that is neither INT-positive nor culturable and reinforce previous results that still mineral waters have a large bacterial population after storage.Key words: mineral water, bacterial populations, ethidium bromide, INT, HPC.