Estimation of somatic cells
The somatic cells were counted microscopically (Gera et al., 2011). Briefly: Following thorough mixing of milk samples and with a 4 mm diameter platinum loop, 0.01 ml of milk was spread evenly over four 1.0 cm2 area template outlines.
Slides were stained for 30 s in Newman-Lampert stain, the composition as follows:
Methylene blue : 1.2 gm
95% ethyl alcohol : 54 ml
Tetrachloroethane : 40 ml
Glacial acetic acid : 6 ml
Somatic cells were stained with deep blue nuclei against light blue background.
The working factor of the microscope was calculated to be 35,400 by using stage micrometer, calculating diameter of the microscopic field (0.012 cm) and field per square cm (8,850) for the given microscope.
Total no. of cells was obtained by multiplying the total no. of cells counted in 25 fields with the working factor.