The objective of this study was to improve Niven's medium (NM) for the optimized enumeration of histamine-forming bacteria (HFB). The parameters modified related to solidification of the agar at low pH values (pH 5.3 to 5.8), incubation time (24, 48, and 72 h) and temperature (30 and 37°C), number of colonies developed on the plate to allow enumeration of HFB, and color differentiation. Strains of HFB, Morganella morganii, Klebsiella pneumoniae, and Hafnia alvei were examined for their ability to change color on NM. The three microorganisms produced different colors on the medium, which can be used for preliminary identification of HFB. Quantitative analysis of HFB proved to be achievable, with the prerequisite that only 1 to 80 colonies developed on the medium allow effective enumeration. A larger number of colonies results in color development throughout the medium, making the distinction between HFB and other bacteria unachievable. Growth of prolific HFB was noticeably better at pH values from 5.3 to 5.5, compared to 6.3, on NM. Growth at 5.3 and 5.5 on NM also presented a significant advantage in comparison to growth on plate count agar (PCA; pH 7) at the same incubation temperature. The increased agar concentration of 3% was found to give better solidification at pH 5.3 to 6.0, compared to 2%. This agar concentration also allows autoclaving for 12 min at 121°C, overcoming the hydrolysis problems that appear at the lower concentration of 2%. The construction of a color chart for the recognition of the pH change due to histidine decarboxylase activity was also achieved.

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