Bat Leukocyte Enumeration and Validation

Blood smears were stained with May-Gruenwald’s solution (#T863.2, Carl Roth GmbH) and Giemsa (#T862.1, Carl Roth GmbH). Total WBC count for each individual was estimated manually by taking the mean of 10 visual fields, counting the cells with a microscope under 200× magnifications. Some former publications using the same method multiplied this mean with a certain species-specific constant to obtain the number of white blood cells per microliter (e.g. [31] (link), [32] (link)). As no such constant is known for bats yet, and multiplication would not change the relative differences among species, we used the mean number of leukocytes per visual field for statistical analysis.
Additionally, after estimating the total WBC counts from blood smears, we validated this method by checking for correlation between our data obtained from blood smears and data obtained with a conventional method (Unopette™ capillary system) of the same species at the same site in a previous year [30] (link), finding a strong positive correlation (linear model; R² = 0.803; N = 12; t = 7.06; p<0.001).
Differential white blood cell (DWBC) counts were performed counting 100 leukocytes under 1000× magnification (oil immersion) and calculating the relative numbers of lymphocytes, monocytes, neutrophils, basophils and eosinophils. Absolute numbers of the different leukocytes were calculated by multiplication with total WBC counts.