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-use open-type hemocytometer w/ Improved Neubauer ruling
→ Spencer Brightline Double Counting Chamber w/ Improved Neubauer Ruling

Fig 1.Illustration of the Hemocytometer

*depth-distance from or between the hemocytometer and cover slip

-0.1mm or 1/10 for Improved Neubauer Ruling
-part of the factor

Fig 2. The Ruled Area of Hemocytometer (Neubauer)

There are 9 large (1mm) squares. Each of the 4 corner 1mm squares subdivide into 16 smaller squares and is marked W
to indicate use for counting leukocytes. The center 1mm sqyare is subdivided into 25 smaller squares each 0.4mm2. The
squares marked R were used for counting RBC manually. The higher the magnification of the center square illustrates all
25 0.04mm2 squares marked P where platelets are counted
B. Coverslip
-magnifies specimen-more magnified cells
-has special optical property thus expensive
C. Thoma diluting pipet or Unopette’
-Thoma pipets used to dilute specimens for all counts. Upper pipet is used to mke 1:20 or 1:10 dilution.
Lower pipet is used to make 1:100 or 1:200 dilution for elevated leukocyte counts; this pipette was historically used
for manual RBC Cts w/c are no longer performed

▪ Unopette
-common diluting pipet
a. Bulb: larger bulb for RBC pipets to hold 100 volumes
Smaller bulb for WBC pipets to hold 10 volumes
b. Beads for mixing identification
c. Calibration mark
RBC: 101
WBC: 11

D. Pipette Suction Apparatus –resembles sucking tube w/ mouthpiece

E. Diluent for Leukocyte Counting
-must be HYPOTONIC to lyse red cells
o Glacial Acetic Acid 2%v/v =nearly 100% concentration
-3mL HAc + 1mL 1% gentian violet w/v, dilute w/ dist H2O to 100mL
o HCl 1%v/v
-3mL HCl + 1mL 1% gentian violet w/v, dilute w/ dist H2O to 100mL
o Turk Solution
-has the stain Gentian violet incorporated in it which stains the nucleus of the WBC
-5% Turk solution