Hemocytometer Measurements - (Oct/11/2008 )

Hi,

I have to perform a practical using hemocytometer.

I cannot understand who to use it even using various protocols including some protocols of Monica.

SO some one please tell me

the procedure of complete blood count. which shoud be easy


thanx

---

The haemocytometer is a device originally used to count blood cells (as the name suggests). It is now also used to count other cells and many types of microscopic particles. It consists of a thick glass microscope slide with a rectangular indentation that creates a chamber of certain dimensions. This chamber is etched with a grid of perpendicular lines.

The raised edges of the haemocytometer hold the coverslip 0.1 mm off the marked grid. This gives each square a well defined volume. The device is carefully crafted so that the area bounded by the lines is known, and the depth of the chamber is also known. Therefore it is possible to count the number of cells in a specific volume of fluid, and thereby calculate the concentration of cells in the fluid overall.

When a liquid sample containing immobilized cells is placed on the chamber, it is covered with a cover glass, and capillary action completely fills the chamber with the sample. Looking at the chamber through a microscope, the number of cells in the chamber can be determined by counting. Different kinds of cells can be counted separately as long as they are visually distinguishable. The number of cells in the chamber is used to calculate the concentration or density of the cells in the mixture from which the sample was taken: it is the number of cells in the chamber divided by the chamber's volume (the chamber's volume is known from the start), taking account of any dilutions

Tips when using a haemocytometer
• Mix your original mixture thoroughly before taking a sample.

• Use an appropriate dilution of your mixture with regard to the number of cells you hope to count. If your sample is not diluted enough, the cells will be too crowded and difficult to count. If it is too dilute, your sample size will not be enough

• Analyze multiple sections. By performing a second or third etc. test on a further chambers, you can compare the results and remove any discrepancies

• Do not use paper towel to dry the excess liquid. The same capillary action that filled the chamber will then dry it out.

• Watch out for the objective lens. Remember that the haemocytometer is thicker than a normal microscope slide. If you focus too closely, your objective lens may contact the instrument

• You don't have to count the whole chamber. If there are alot of cells, you can just perform your count in a section of the chamber and use the grid to determine what proportion of the chamber that is. You can then extrapolate to estimate correct no

Methodology
Counting red cells by visual means
1. Make 1:200 dilution of blood in buffered solution (20μl blood in 4 ml diluting fluid) - possibly PBS
2. Mix blood solution by inversion for 2 minutes
3. Add 20μl of this blood solution to a haemocytometer with cover-slip (this is attached to haemocytometer by breathing on the coverslip and pressing it onto the slide)
4. Examine microscopically using 10x or 40x lens
5. Count cells on 5 random inner squares within the central big square

Result:
Red-cell count per/mL:

((number counted in 5 inner squares divided by 0.02) X 1,000) X by dilution factor) which is 200 in this case as we made a 1:200 dilution with PBS so say your total count is 150, then (150 / 0.02) x 1,000 = 75,000 x dilution factor = 200 giving 15,000,000 cells /ml of blood

repeat these steps a couple more times taking a new sample from the dilution each time and then take the average of these results to get a more accurate result.

---