Methods of calculating total hemocyte count in Australian red-claw crayfish

Abstract

The optimal formula for calculating the total number of hemocytes (HTN) of the Australian red-claw crayfish (Cherax quadricarinatus) has been established, which allows optimizing the time of counting HTN without re-ducing accuracy. Methodical recommendations on preparing a hemolymph sample, HTN calculation and differentiated calculation of hemocytes are given. It was found out that the differences in HTN are minimal when using different formulas, which is con-firmed by the analysis of variance F = 0.011, p = 0.998, the average values of HTN range from 2 384 to 2 427 cell/µl. Conducting multiple comparisons also demonstrated that the differences between the formulas are minimal: the significance levels between the formulas were p = 0.99. The optimal formula for the Australian red-claw crayfish is: HTN in 1 µl = N • 10, where N is the total number of hemocytes in 25 large squares of Gorjaev’s chamber. The counting chamber should be clean, since when even small dust particles are found on the grid, hemocyte clusters can form and impede counting, or the result of the calculation will be incorrect, because the distribution 
 of hemocytes on the grid will not be uniform. After filling the chamber with hemolymph, it is recommended to wait 
 1-1.5 minutes so that the movement of the hemolymph stops and the hemocytes stop moving. It is recommended to use an anticoagulant, which prevents the rapid decay of cells and promotes their uniform distribution on the grid of the chamber without developing clots (clusters) that affect the accuracy of counting. Working with the whole hemolymph implies the speed and accuracy of the researcher's actions, since it quickly coagulates and forms clusters of hemocytes. In order to avoid confusion when counting cells located on the border between squares, Egorov’s rule is applied. The optimal increase for counting is a 400-fold increase.