Total Leukocyte Count | TLC Blood Test Procedure | Total WBC Count Experiment

This video explains the procedure of Total Leukocyte Count ( #tlctest or TLC Blood Test ) using Neubauer Chamber or Haemocytometer with calculations. It is also known as Total WBC Count Experiment, White Blood Cell Total Count, TLC lab test, TLC DLC blood test, etc. In Hindi सफेद रक्त कोशिका जांच,
tlc test kaise karte hai?
For detailed information visit: https://www.ebiologylab.com/experimen...
Principle of TLC Blood Test:
The total number of leukocytes WBC per mm3 of blood is determined by diluting the known volume of blood 20 times with fluid, which prevents hemolysis and coagulation. The blood cells are then counted in the volume of diluted blood in Neubauer’s counting chamber or Haemocytometer. The number of WBCS per mm3 of the undiluted blood is then determined by this method.
Apparatus required for #TLCBloodTest:
1.) Neubauer Blood Counting Chamber: https://amzn.to/2S9VpR2
2) WBC Pipette: https://amzn.to/3xutNoD
3) WBC diluting fluid or Turk’s fluid: https://amzn.to/3q4yJ0W
4) Compound microscope: https://amzn.to/2U6Lj3S
5) 90% ethyl alcohol: https://amzn.to/3xsJpZS
6) Sterile cotton: https://amzn.to/3iOKdEa
7) Sterile needle: https://amzn.to/2U6LuMA
8) Reagent Bottles: https://amzn.to/3xvRBZj

Neubauer’s hemocytometer:
The instrument is consists of a special glass slide. The counting chamber is a very heavy thick glass slide usually at the center with three platforms, which is separated by wide grooves. The central platform is 0.1 mm lower than its neighbor. On the surface of the middle platform, there are two sets of ruled areas. The area has 9 large squares, each has 1 square millimeter area and is elaborately ruled. The four corner squares are divided into 16 equal-sized squares. The central square is ruled into 25 groups of 16 small squares, each group is separated by triple lines. The ruled surface area is 1/10 millimeter below the inner surface of the cover glass placed over the middle platform. Thus each smallest square of the corner has a volume of 1/4 x 1/4 x 1/10 or 1/160 mm3. Whereas the smallest square in the center has a volume of 1/20 x 1/20 x 1/10 = 1/4000 mm3. This central segment is used for RBC counting. In four big squares, leukocytes or WBCs is are counted which are placed at the four corners of the erythrocyte counting Chambers.
WBC diluting fluid or Turk’s fluid: https://amzn.to/3q4yJ0W
The fluid consists of Glacial Acetic Acid, 1.5 ml (for destruction and dissolution of RBCs and to fix WBCs); Gentian violet solution, 1.5 ml (1% solution, stain WBCs); these components are mixed and diluted 100 ml by distilled water.
The procedure of TLC Blood Test:
1. The counting chamber is placed on the microscope stage. The illumination is adjusted and the right upper groups of 16 squares for WBCs are focused. One can see all the squares in the alone field.
2. 1 ml of Turk’s fluid is taken in a watch glass.
3. Collection of blood:
The tip of the finger is sterilized with 90% alcohol and allowed to dry. The finger is pr.cked with a needle to produce a drop of blood of adequate size 3 to 4 millimeters in diameter. The blood is sucked up to the 0.5 mark into the WBC pipette.
4. The blood is diluted with Turk's fluid up to 11 mark by sucking the diluting fluid by the pipette kept in a watch glass. When the blood is taken up to the mark of 0.5 the blood is diluted only 20 times. The contents inside the bulb of the pipette is mixed for 3 to 4 minutes.
5. The first two drops of diluted blood from the Pipette are discarded and then this mixture is run on the hemocytometer slide on both the chambers on both sides under a special coverslip.
6. The corpuscles are allowed to settle down for 3 to 4 minutes and then the number of WBCs are counted under the low power of the microscope in the four counting areas.
7. The white cells are recognized by the refractile appearance and by the slight color given to them by the stain contained in the diluting fluid.
8. The cells are counted under high-power lens. One may count the WBCs in 16 squares under low power and then under high power for comparing the results. The cells in four groups of 16 squares each i.e., a total of 64 squares.

Counting procedure: Cells which are touching the left-hand lines or upper lines of the square are included in the count, while cells touching the lower and right margins are excluded.


Haemocytometer Calculation:
The WBCs counted in 16 squares the volume of one square being 1/64 mm3
Volume of 16 square = 1/160 x 64 = 4/10 mm3
Thus, the total volume of diluted blood in which WBCs were counted = 4 / 10 mm3
Let the count in 4/10 mm3 = ‘X’ WBCs.
Then 1 mm3 of diluted blood will contain = ‘X’ x 10/4 white cells of WBCs.
As dilution is 20 times, 1 mm3 of undiluted blood will contain = ‘X’ x 10/4 x 20 = ‘X’ x 200/4 = ‘X’ x 50 WBCs. In general, 1 mm3 of undiluted blood will contain = ‘X’ x 10/4 x dilution factor of WBCs where, dilution factor maybe 20 or 10.