Total RBC Count Practical Lab

Check out my other Haematology videos! Click on HASHTAGS above this video and the links given below to watch my other related videos.
Total WBC Count: https://www.youtube.com/watch?v=xyhbIPSLBsA&t=364s
Total RBC Count: https://www.youtube.com/watch?v=0f9p9JX4qJk&t=130s
DLC: https://www.youtube.com/watch?v=VFKm_kMTf50
Haemocytometer Use: https://www.youtube.com/watch?v=7IpE33WPRrc&t=43s
Haemometer Hb estimation: https://www.youtube.com/watch?v=6CqptdZyUaU&t=45s
Blood Smear Prep and Staining: https://www.youtube.com/watch?v=KSs0SMfERuA&t=38s
RBCs in iso, hypo and hypertonic solns: https://www.youtube.com/watch?v=urYJAPGUpuw
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RBC Count
In this video, we’re gonna count the number of red blood cells in a sample of human/animal blood using the haemocytometer.
Requirements:
Counting chamber
RBC pipette
Blood sample
RBC diluting solution
(Hayem’s fluid)
Mercuric chloride 0.5g (acts as antiseptic)
Sodium Chloride 1.0g (provides isotonic environment)
Sodium sulphate 5.0g (prevents RBC roleaux formation)
Distilled water 200ml (Solvent)
Compound light microscope
Cotton and Rubbing alcohol

Take the RBC pipette precleaned with alcohol.
Draw the blood sample till the 0.5 mark using your mouth or a syringe, while taking care to avoid any air bubbles from entering the pipette.
This step requires a bit of patience and practice. If you happen to suck the blood beyond the 0.5 mark, you can either use a clean filter paper to carefully blot out excess blood from the pipette tip, or carefully blow out excess blood using a syringe until it reaches down to the 0.5 mark.
Once that’s done, carefully clean the excess blood sticking to the outer sides of the tip of the pipette using a cotton soaked in alcohol.
Now carefully draw the RBC upto 101 mark on the pipette.
Hold the pipette in a horizontal position and Rotate the pipette several times in such a way that the tiny bead inside the bulb of the pipette mixes the blood and the diluting solution thoroughly.
Once thorough mixing has been accomplished, the blood sample now has a 200 times dilution.
Once this is done, prepare the counting chamber for sample loading.
Take the counting chamber and the cover slip, both precleaned with alcohol.
Carefully position the coverglass on top of the support platforms located on both sides of the counting chamber platforms.
Discard the first few drops from the RBC pipette. Carefully position the tip of the pipette on one edge of the coverglass.
Allow the contents of the pipette to gradually flow into the narrow space between the coverglass and the counting chamber via a capillary force like you see here. You may load the other counting chamber as well in a similar fashion.
Once ur done loading the chambers, keep aside the counting chamber for a few minutes to allow the RBCs to settle.
The slide is now ready for microscopic observation.
First locate the small square at the centre of the counting grid under low power of the microscope. First locate the large squares at the four corners of the counting grid under low power of the microscope. You may refer to my previous video on Haemocytometer to see which of the squares in the counting chamber are to be used for RBC counting.
You may also need to adjust the light intensity of the microscope in order to visualise the fine lines in the counting grid.
Once you’ve located the RBC counting squares, switch to 10x then to 40x objective to begin the counting.
Locate the 1st, 5th, 13th, 21st and 25th smaller squares in the RBC grid.
Applying the Margin Rule or L rule of cell counting, Count the RBCs lying inside of these squares and those lying on the borders of the lower and left sides of each square. Do not count those cells lying on the borders of the upper and right sides of the squares.
Remember the margin rule and count the cells in each square accordingly.

Calculation:
Once you’ve counted the number of cells in the… 5 smaller squares aka the 80 smallest squares, all you have to do is to just add them up and multiply the sum by a factor of 10,000 This gives you the total RBC count per cubic mm of blood sample.
Or if you wanna do the math, you can use this formula to come up with the number of RBCs in 1 cubic mm of the blood sample.
RBC count = No of cells counted x Dilution factor x Depth factor/ Area counted
We know that:
Dilution of the blood sample is 1 in 200. So, Dilution factor is 200.
Depth or Height of Blood film is 1/10th mm. Therefore Depth Factor is 10
Area counted is 80 /400 smallest squares = 1/5 sq mm
Assuming that the no of cells counted is “N” and using the above known data,
The total RBC count per cubic mm of blood= N x 200 x 10/1/5th
= N x 10,000

Видео Total RBC Count Practical Lab канала ThomasTKtungnung