Hemocytometer with its principle, content and application.

1. HEMOCYTOMETER
D r. A niket A . Shilwa nt
Assistant Professor
Sharir Kriya Dept.
GJPIASR, CVM University

2. What is Hemocytometry ?
It is a technique used to enumerate the total cell
count in the BLOOD or any other biological fluids.
It can be done using Hemocytometer manually or by
Electronic cell counter automatically.
Hemo – Blood
Cyto – Cell
Meter - Measurement

3. Principle of Hemocytometry
 Since the number of blood cells is very high so it is highly impossible to
count them by naked eyes.
 It is overcome by magnification upto some extent and can be corrected by
proper dilution.
 For this the sample of blood is diluted to a known degree with suitable
diluting fluids and later counting can be done.
 The sample of blood is first diluted in a special volumetric pipette and the
drop of a diluted solution is then placed under cover slip placed over a
special thick glass slide having counting grid over it.
 Knowing the dilution factor applied, the number of cells in undiluted blood
can be calculated and thus the final result so obtained is expressed as cells
per cubic mm.

4. Purpose of Hemocytometry
 To find out the normal or abnormal count so as to know the functional
efficacy (Haemopoeisis) of marrow status.
 To support and confirm the clinical diagnosis of the subject.

5. SET OF HEMOCYTOMETER –
It consists of –
1. Neubauer’s counting chamber
2. A total two Volumetric diluting pipettes
(RBC & WBC)
3. Cover slip
4. Associated with –
Two diluting fluids for - RBC & WBC

6. H E M O C Y TO M E T E R

7. HEMOCYTOMETER
Inventor – Louis Charles Malassez
Improved and Modified by – Neubauer
Structure (Counting chamber) –
Special thick glass slide
Having H shaped platform with trenches forming
H shape.
Have counting grid over and below the horizontal
line of H shape designed for counting of cells.
The H shape platform is slightly (0.1mm) below the
actual surface of glass slide. This gives the depth of
the chamber when it is covered with cover slip.

8. W W
R
W W
W W
R
W W
Depth
0.1mm

9. Counting grid –
• Each grid is dividend of 3mm wide and 3mm
long making a square with area – 9mm2
• The complete grid is of 9 small squares of 1mm
each with area – 1mm2
• The four corner squares are further divided into
16 small squares.
• A total of 64 small squares present on four
corners specially made for WBC counting.
• A central large square is divided into 25 small
squares. Each small square is further dividend of
16 smallest square within it.
• A total of 25 small squares or a total of 400
(25 x 16) smallest squares centrally each one etc
hed by triple line is specially made for RBC
counting.

11. CALCULATION OF VOLUME OF WBC SQUARES
 WBC = 4 CORNER SQUARES
 Length of 1Corner Square = 1mm
 Area of 1Corner Square = 1mm2
 Area of 4Corner Square = 4mm2
 For Volume, 3rd side = Height or Depth
 Depth of Chamber = 1/10mm or 0.1mm
 Volume of 1Corner Square = 1mm3
 Total Volume of diluted blood in 4corner squares
= 4mm2x 1/10mm
= 4/10mm3

12. WBC 4x4 = 16
16 X 4 = 64

13. CALCULATION OF VOLUME OF RBC SQUARE
 RBC = CENTRAL SQUARES
 Length of Central Square = 1mm (Divided into 25 small squares)
 Length of 1 small square = 1/5mm (Divided into 16 smallest squares)
 Length of 1 smallest square = 1/5 x ¼ = 1/20mm
 Area of 1 smallest square = 1/20mm x 1/20mm = 1/400mm2
 For Volume, 3rd side = Height or Depth
 Depth of Chamber = 1/10mm or 0.1mm
 Volume of 1 smallest square = 1/400mm2 x 1/10mm = 1/4000mm3
 Total Volume of diluted blood in 80smallest square (5small = 16 x 5)
= 80 x 1/4000mm3 = 1/50mm3

14. R1 R2
R5
R3 R4
RBC
5X5 = 25
16 X 5 = 80

15. C O U N T I N G R U L E

16. Rule No. 1 – L Rule or Inverted L Rule
• Cells present on left vertical and below horizontal
line (which makes L for every square) should be
counted within the same square. While cells on
right vertical and above horizontal line are to be
counted in adjacent squares.
Rule No. 2 – Triple line Rule
• Cells present on the very 1st line which contribute
to make a square are to considered for counting
in a square and not those cells which are present
on 2nd or 3rd line.
• Cells present on 1st and 2nd line but not intruding
within the square are not considered for counting.
Considered
Not Considered

17. Rule No. 3 – Zigzag Rule
• Always count the cells in Zigzag manner either
from left to right or vice versa.

18. D I L U T I N G P I P E T T E S

20. Sr.
No
Difference in
Parameters
RBC Pipette WBC Pipette
1 Bead Red White
2 Markings 0.5, 1.0, 101 0.5, 1.0, 11
3 Size of bulb Larger Smaller
4 Mouth piece color Red White
5 Dilution 100 / 200 times 10 / 20 times
6 Bore Diameter Narrow Wide
DIFFERENCE BETWEEN RBC & WBC PIPETTE

21. C H A R G I N G O F C H A M B E R

23. D I L U T I N G F L U I D S

24. R B C D I L U T I N G F L U I D
Sr. No Content Concentration
A. HAYEM’S DILUTING FLUID
1 Sodium Chloride (NaCl) 0.50 gm
2 Sodium Sulphate (Na2SO4) 2.50 gm
3 Mercuric Chloride (HgCl2) 0.25 gm
4 Distilled Water 100 ml
B. DACIE’S SOLUTION
1 Tri-Sodium Citrate 3.13gm
2 37% Formalin 1ml
3 Distilled Water 100ml
C. NORMAL SALINE – 0.85 to 0.90%

25. W B C D I L U T I N G F L U I D
Sr. No Content Concentration
TURK’S Diluting Fluid
1 Glacial Acetic Acid 1.5 ml
2 Gentian Violet Solution 1.5 ml
3 Distilled Water 100 ml

27. Thank You All !!!
Dr. Aniket A. Shilwant
Assistant Professor
Department of Kriya Sharir
GJP-IASR, CVM University
Email – ayuraniket18@gmail.com
http://ayugjac.edu.in/Staff_CV.aspx?dl=dn3Mja19480dn3Mja19
http://scholar.google.co.in/citations?user=636K2sMAAAAJ&hl=en
https://www.researchgate.net/profile/Aniket_Shilwant