A. Hemocytometer-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-parts:

a. Bulb: larger bulb for RBC pipets to hold 100 volumesSmaller bulb for WBC pipets to hold 10 volumes

b. Beads for mixing identificationc. Calibration mark

RBC: 101WBC: 11

D. Pipette Suction Apparatus –resembles sucking tube w/ mouthpieceE. Diluent for Leukocyte Counting

-must be HYPOTONIC to lyse red cellsExamples:

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