Data associated with the characterization and presumptive identification of Bacillus and related species isolated from honey samples by using HiCrome Bacillus agar

The dataset described in this paper provides information on the morphological features of 24 different species of the genera Bacillus, Paenibacillus, Brevibacillus, Lysinibacillus, and Rummeliibacilluswhen growing in HiCrome Bacillus agar. The species studied are common contaminants of honey. In support to the recent publication entitled “HiCrome Bacillus agar for presumptive identification of Bacillus and related species isolated from honey samples” (2), a collection of 197 bacterial isolates belonging to 24 different species of aerobic spore-forming bacteria have been screened for their colony appearance and color and any substrate color change of HiCrome Bacillus agar at 24 and 48 h of incubation. Two simple flowcharts utilizing a combination of colony and media characteristics in the chromogenic medium and a set of simple biochemical and morphological tests were developed for quick presumptive identification.


Data
The dataset described in this paper provides information on the morphological features of 24 different species of the genera Bacillus, Paenibacillus, Brevibacillus, Lysinibacillus, and Rummeliibacillus when growing in HiCrome Bacillus agar. The species studied here have been previously reported in honey [1e3, 5,7,10,12e15].
A collection of 197 bacterial isolates belonging to the 24 species tested have been screened for their colony appearance and color and any substrate color change of HiCrome Bacillus agar at 24 and 48 h of incubation ( Fig. 1 and Table 3). Colors of colonies and substrate observed were compared with a Pantone international chart and identified with a PMS number (http://www.cal-print.com/ InkColorChart.htm).
The Ecometric technique was used for comparative evaluation of HiCrome Bacillus agar and the control medium (Figs. 2 and 3 and Table 3). E-values (Table 3) were obtained for 28 selected isolates tested by using previously published methods [2,8].
Two Flowcharts were prepared by a combination of colony and media characteristics in HiCrome Bacillus agar and a set of selected biochemical and morphological tests that are used routinely in Microbiological laboratories. The first chart (Fig. 4) permits the identification of the aerobic sporeforming bacteria reported in honey by a few simple tests. The more simplified flowchart presented in Fig. 5 allows differentiating typical strains of aerobic spore-forming species by direct isolation from honey.
The bacterial identity of selected strains isolated from honey or honeybee larvae (n ¼ 56) ( Value of the data Data presented to describe the colony appearance on HiCrome Bacillus agar of several Bacillus and related species commonly found in honey. Data generated serve as a point of reference for further research in microbial diversity, microbial ecology, and microbial taxonomy.
The information presented could be beneficial for other researchers that are interested in the microbiology of honey and is potentially also of benefit for research on other food products.

Experimental design, materials, and methods
A collection of 197 bacterial isolates of Bacillus, Brevibacillus, Lysinibacillus, Paenibacillus, and Rummeliibacillus belonging to different species that have been reported in honey [1e3,5,7,10,12e15] were screened for their abilities to grow and colony appearance and color, and any substrate color change by using HiCrome Bacillus agar. The collection includes 167 isolates from honey samples from different geographical areas including Argentina, Brazil, France, Italy, and Mexico; 9 isolates from honeybee larvae from different geographical areas including Argentina, France and Sweden; and 21 strains from Culture Collections used for comparison and quality control. Bacteria were maintained as stock cultures at À80 C in the correspondent broth medium, Müller-Hinton Broth, yeast extract, potassium phosphate, glucose, and pyruvate (MYPGP) [4] or Brain heart infusion (BHI) plus 20% glycerol (v/v). For short-term storage, the strains were kept at 4 C in screw-capped vials containing MYPGP or BHI semi-solid (0.4% agar).  Bacterial smears stained by Schaeffer-Fulton technique were examined for the presence and location of spores within cells, as well as for the size and shape of vegetative cells [9,11]. Also, the presence of unstained globules in the cytoplasm [6,9,11] was examined by phase contrast microscopy (Leica, ICC50).
The Ecometric technique was used for comparative evaluation of HiCrome Bacillus agar and the correspondent control media (BHI or MYPGP). Overnight cultures were adjusted to 0.5 Mc Farland in sterile distilled water. One loop of 10 ml of each suspension was sequentially diluted from streak to streak onto each medium by inoculating 21 streaks (5 per quadrant and 1 in the center). Growth on the plates was recorded as a score. Readings were presented as absolute growth indices with possible values of 0e5, where 0 is an absence of growth in any streak and 5 was the maximum score obtained when all of the streaks in the four quadrants and also the last streak showed visible bacterial growth [2,8]. Twenty-eight bacterial strains with different colony types ( Table 2) were used for the evaluation. Plates were inoculated and incubated in duplicate for 24e48 h at 37 C. Scores for HiCrome and control plates were compared to estimate the degree of inhibition due to the chromogenic mixture (Table 3, Figs. 2 and 3).
For purification of PCR products the following enzymatic procedure was used: The mixture con- The quality and quantity of PCR products were assessed by gel electrophoresis (1 ml/1.6% agarose/ molecular weight marker QuantiMarker, Promega, Argentina) and DNA concentration was estimated