Hemocytometer

The process of counting cell using Neubauer’s Slide

1. Hemocytometer Prepared by B.S Physiology Semester 1 Group 4 Asad Ali Sidre Abbas Sundas Tabinda Maryam Mumtaz Amina jameel Ahsan Hayat

2. Key points to know What is hemocytometer? Uses of hemocytometer? Principle of cell counting using Neubauer's slides Common sources of error during the process of Hemocytometery

3. Hemocytometer Hemo : Blood Cyto : Cell Meter : Measurement/Counting Thus , it is a device originally designed and usually used for counting blood cells.

4. Material required Microscope Neubauer’s slides Cover slip Cover glass Diluting pipette Cell suspension Trypan blue

5. Neubauer’s Slides Thick glass slides with two ruling areas on center Ruling areas are separated by H-shaped trough. Beyond trough, there are two raised arms to support cover glass lining is coated by shiny metal or rhodium. Neubauer’s slide with a cover slip over it is called Neubauer’s chamber.

6. Each scale is 3mm wide and 3mm long. Depth of the chamber is 0.1mm. The whole scale is divided into 9 big squares. Each square is 1mm long and 1mm wide.

7. The four corner squares are further divided into 16 smaller squares and are used for WBC counting. Total 64 small squares are there which have a dimension of 0.25mm x 0.25mm = 0.0625mm

8. Central square is divided into 25 medium sized square and are separated by triple line The medium sized square are further divided into 16 small square(tiny) The four corner and central square are used for cell count

9. Cover Slides Special cover glass with smooth surface and even thickness Thickness= 0.3, 0.4, 0.5 mm Length= 16x22mm, 22x23mm

10. Preparing the slide Draw the cell suspension into pipette up to 100 μL Draw 100 μL of Trypan blue (to differentiate live and dead cells) Load both chambers of the Neubauer's slide by pipetting the suspension under the cover slip.

11. Microscopy Now place the Neubauer’s chamber under the microscope. Focus must be 4X to see the general formation of slides 10X for WBC counting 40X for RBC/ platelets counting

12. View at 4X

13. View at 10X

14. At 40X

15. Cell counting Count the cells in 4 corner squares The center square

16. Cell Counting Do not count cells touching Bottom line Right line

17. count Don’t Count

18. Microscopic view of slides to count cells

19. Count cells in the five selected chambers of cell count.

20. 5

21. Calculation 1. Average # of cell per square = Total number of cells / Total squares 2. Dilution factor = final volume/ volume of cell 3. Concentration of cells = average # of cell / dilution factor

22. Applications Blood count : for patients with abnormal blood cells, where automated counters don't perform well. Sperm count:

23. Applications Cell culture: when subculturing or recording cell growth  over time.

24. Possible errors Errors in this process are of two main types False high count False low count

25. False high count Uneven distribution of cell Improper mixing Error in pipetting Error in calculation Blood taken from area of hemo concentration Yeast, dirt and leukocyte are counted as RBC

26. False low count Blood diluted with tissue fluid Undue delay in counting of cell Clumping of cell(AIHA) Uneven distribution of cell Faulty technique of counting Improperly standardized counting chamber