RAPID COUNTING OF ACETIC ACID BACTERIA BY THE EXCLUSION METHOD USING TRYPAN BLUE UNDER NEUBAUER’S CHAMBER

Acetic acid bacteria (BAA) play an important role in the production of vinegar and are responsible for the oxidation of dilute ethanol to acetic acid and water. Rapid and accurate quantifications of viable BAA cells are of great interest to the vinegar industry since its process is dynamic and requires quite strict control. Conventional detection methods such as the plate counting method, besides requiring a longer time, may result in underestimation of the microbial concentration, although this problem of isolation and cultivation has been partially reduced by the introduction of the double-layer agar technique (0,5% agar in the bottom layer and 1% agar in the top layer). Thus, the objective of this study was to propose the exclusion test with trypan blue (TB) under the Neubauer chamber as an alternative and faster method instead of the conventional method. The strain Komagataeibacter intermedius, isolated from vinegar production was obtained from the bacterial collection of the Laboratory of Food Analysis of the Universidade Estadual of Londrina (UEL). Primarily, the bacterial isolate was cultured (30 °C/72 hours/120 rpm) in MYP (Manitol, Yeast, Peptone) broth and the standardization of the inoculum was performed by spectrophotometry at 580 nm (% T 22-26), so that counts close to 9 log CFU/mL were obtained. This inoculum was then added with TB dye (0,2%) for counting of Neubauer chambers and subsequent analysis under an optical microscope. At the same time, counting was done by the droplet-plate method containing MYP using the double-layer technique to compare the two counting methods. Our results showed that the cellular viability of K. intermedius, using the TB exclusion test and the plaque count showed no significant difference between them, with counts of 8,64 ± 0,19 log CFU / mL and 9,0 ± 0,15 CFU / mL, respectively. Therefore, the use of the TB method under Neubauer chamber can be considered promising in the control of the microbiological quality of vinegar industries.