Demonstration Cred Blood Cell Count

Although modern clinical laboratories commonly use electronic instruments to obtain blood cell counts, a hemocy-tometer provides a convenient and inexpensive way to count both red and white blood cells. This instrument is a

Figure 39.1 Steps in the red blood cell percentage procedure: (a) load a heparinized capillary tube with blood; (b) plug the blood end of the tube with sealing clay; (c) place the tube in a microhematocrit centrifuge.

Figure 39.1 Steps in the red blood cell percentage procedure: (a) load a heparinized capillary tube with blood; (b) plug the blood end of the tube with sealing clay; (c) place the tube in a microhematocrit centrifuge.

Stomata Count Grid CoverslipMicrohematocrit Procedure Grid Slide Coverslip

special microscope slide on which there are two counting areas (fig. 39.3). Each counting area contains a grid of tiny lines forming nine large squares that are further subdivided into smaller squares (fig. 39.4).

The squares on the counting area have known dimensions. When a coverslip is placed over the area, it is held a known distance above the grid by glass ridges of the hemocytometer. Thus, when a liquid is placed under the coverslip, the volume of liquid covering each part of the grid can be calculated. Also, if the number of blood cells in a tiny volume of blood can be counted, it is possible to calculate the number that must be present in any larger volume.

When red blood cells are counted using a hemocy-tometer, a small sample of blood is drawn into a special pipette, and the blood is diluted to reduce the number of cells that must be counted. Some of the diluted blood is spread over the counting area, and with the aid of a microscope, all of the cells in the grid areas marked R are counted (fig. 39.4). The total count is multiplied by 10,000 to correct for the dilution and for the fact that only a small volume of blood was observed. The final result provides the number of red blood cells per cubic millimeter in the original blood sample.

1. To perform a red blood cell count, follow these steps: a. Examine a hemocytometer, and use a microscope to locate the grid of a counting area. Focus on the large central square of the grid with low-power and then with high-power

Figure 39.2 Steps in the hemoglobin content procedure: (a) Load the blood chamber with blood; (b) stir the blood with a hemolysis applicator; (c) place the blood chamber in the slot of the hemoglobinometer; (d) match the colors in the green area by moving the slide on the side of the instrument.

Figure 39.2 Steps in the hemoglobin content procedure: (a) Load the blood chamber with blood; (b) stir the blood with a hemolysis applicator; (c) place the blood chamber in the slot of the hemoglobinometer; (d) match the colors in the green area by moving the slide on the side of the instrument.

Pic Wbc HemocytometerWbc Count With HemacytometerPic Wbc Hemocytometer

Figure 39.3 Location of the counting areas of a hemocytometer.

Pic Wbc Hemocytometer
Figure 39.4 The pattern of lines in a counting area of a hemocytometer. The squares marked with R are used to count red blood cells, whereas the squares marked with W are used to count white blood cells.
Pic Wbc Hemocytometer
Figure 39.5 The Unopette system consists of a reservoir containing blood cell diluting fluid (left) and a plastic capillary tube within a plastic shield (right).
Pic Wbc Hemocytometer

magnification. Adjust the light intensity so that the lines are clear and sharp.

b. Wash the hemocytometer with soap and water, and dry it. Place the coverslip over the counting areas and set the instrument aside.

c. Examine the Unopette system for counting red blood cells, which consists of a reservoir containing blood cell diluting fluid and a plastic capillary tube assembly within a plastic shield (fig. 39.5).

d. Place the Unopette reservoir on the table and gently force the pointed tip of the capillary tube shield through the thin diaphragm at the top of the reservoir.

Lance the tip of a finger, as before, to obtain a drop of blood.

Remove the Unopette capillary tube from its shield and, holding the tube horizontally, touch the tip of the tube to the drop of blood. Allow the tube to fill completely with blood by capillary action (fig. 39.6). Gently squeeze the reservoir, taking care not to expel any of its fluid content, and while maintaining pressure on the sides of the reservoir, insert the capillary tube through the punctured diaphragm of the reservoir. Release the pressure on the sides of the reservoir and allow the blood to be drawn into its chamber.

Figure 39.6 The Unopette capillary tube fills with blood by capillary action.

Figure 39.6 The Unopette capillary tube fills with blood by capillary action.

Unopette System
Figure 39.7 The Unopette dropper system being used to fill a hemacytometer with blood. By squeezing the reservoir, a drop of diluted blood enters the counting areas under the coverslip by capillary action.
Unopette System

h. Hold the capillary tube assembly in the reservoir and gently squeeze the sides of the reservoir several times to mix the blood with the diluting fluid inside. Also, invert the reservoir a few times to aid this mixing process.

i. Remove the capillary tube assembly from the reservoir and insert the opposite end of the assembly into the top of the reservoir, thus converting the parts into a dropper system (fig. 39.7).

j. Gently squeeze the sides of the reservoir to expel some of the diluted blood from the capillary tube. Discard the first four drops of this mixture and place the next drop of diluted blood at the edge of the coverslip near a counting area of the hemocytometer (fig. 397). If the hemocytometer is properly charged with diluted blood, the space between the counting area and the coverslip will be filled and will lack air bubbles, but no fluid will spill over into the depression on either side. k. Place the hemocytometer on the microscope stage and focus on the large, central square of the counting area with the low-power objective, and then the high-power objective. Adjust the light so that the grid lines and blood cells are clearly visible. l. Count all the cells in the five areas corresponding to those marked with R in figure 39.4. To obtain an accurate count, include cells that are touching the lines at the tops and left sides of the squares, but do not count those touching the bottoms and right sides of the squares. The use of a hand counter facilitates the counting task. m. Multiply the total count by 10,000 and record the result (cells per cubic millimeter of blood) in Part A of the laboratory report.

n. Discard or clean the materials as directed by the laboratory instructor. 2. Complete Part D of the laboratory report.

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Responses

  • j
    What if there are bubbles under the cover slip when counting red blood cells?
    4 years ago

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