1.  Materials: Sputum smear microscopy slides from 12 patients prepared using the Kinyoun acid-fast stain and counterstained with methylene blue solution 

2.  Images acquisition:

2.1 Equipments: a Canon PowerShot A640 -10 megapixels digital camera, attached to a conventional microscope model Zeiss Axioskop 4.

2.2 Number of images: 120 images

2.3 Spatial  Resolution: 2816 x 2112  pixels.

3. Grouping of images:

The images acquired were classified in four groups based on: 1 – background density; 2 – density of bacilli (see figure 2). The  “High Density Background”  group  is characterized  by  a  strong  presence  of  counterstain  with methylene blue solution in the background, while the “Low Density Background” group  is  characterized  by  a  weak  presence  of  this  counterstain.  

Table 1 shows the distribution of images into groups.

4.  Image analysis by a specialist

All acquired images were submitted to a specialist in order to identify the bacilli,   clusters of bacilli and other artifacts  named as doubtful bacilli. Each kind of identified object was surrounded by  a specific geometric shape (see figure 3):

·      A true bacillus was surrounded by a circle.  

·      A cluster of bacilli was surrounded by a rectangle,  and

·      A  doubtful  bacillus  surrounded by  a polygon.

The images with marked objects could be used as gold standard to evaluate the accuracy; sensitivity and specificity of bacilli recognition.