1. 1. MIKROSCOPE And 2.HEMOCYTOMETER
3. ELECTROPHORES 4. pH Meter
Dr.Gatot Ciptadi
Lab. Central of Life Sciences
Fac. Of Animal Sci.
Brawijaya University Malang

2. Counting Micro-organisms , Cells and Sperms
2. Hemocytometer:
Manual, software
Counting Micro-organisms , Cells and Sperms
Counting elephants is easy – counting bacteria is not quite as easy!

3. Counting Micro-organisms
A single tiny drop of nutrient broth incubated overnight may contain cells – this is a lot to count.
1cm3 may contain 108 cells.
In order to estimate numbers it is necessary to dilute the sample.

4. Hemocytometer Chamber
Depth = 0.1 mm
Grids
RBC use 5 small squares in the center large square
WBC use 4 corner large squares

5. Thoma Pipet RBC Aspirate to 0.5 or 1.0 Dilute to 101 DF = 200 or 100
WBC
Aspirate to 0.5 or 1.0
Dilute to 11
DF = 20 or 10

6. The most common routine method for cell counting which is efficient and accurate is with the use of a hemocytometer.

7. Counting Micro-organisms or Sperms
SERIAL DILUTION
1ml into 9ml = 1:10 dilution
conc

9. Counting Micro-organisms
It is possible to dilute a cell suspension in known steps in order to plate a specific range of cells. This set of plates contain a 1 to 10 dilution series.

10. Hemocytometer Originally used in counting RBCs
Consists of a thick glass microscope slide with a rectangular indentation that creates a counting chamber
Counting chamber - engraved with a laser-etched grid of perpendicular lines
Raised edges hold cover slip above these marked grids

11. Cell quantification using hemocytometer
See microscopes on the table.

12. Cell Counting - haemocytometer

13. TOTAL COUNTS Direct counts
The haemocytometer is a specialized microscope slide used to count cells.
The centre portion of the slide has etched grids with precisely spaced lines.

14. Hemocytometer
Counting chamber is a 3 x 3 mm square and divided into nine large squares
Each resulting square measures 1 mm2, which are used for white blood cell counting (Square A)
The center square is divided into 25 smaller squares. (1/25 mm2)
Each smaller square is subdivided into 16 even smaller squares, which are used for counting red blood cells. (Square B = 1/400 mm2)

15. 1 mm
0.1 mm
Volume : 0.1mm3 1 ml = 1 cm3 = 1000 mm3

16. TOTAL COUNTS
To get an accurate count there should be between 40 and 70 cells in a 1 mm square.
If not you dilute or concentrate the cell suspension as necessary .

17. Determination of Number of Cells per mL
Coverslip sits 0.1 mm over the chamber
1mm 3 = 10-3 cubic centimeters (cc) or ml
Volume of a square = 1 mm x 1 mm x 0.1 mm = 0.1 mm3 = 10-4 cc = 10-4 mL
Take the average of the cells found in the 5 squares (the four corners and the middle one).
Avoid counting the cells twice by disregarding the cells found at the upper and left borders
Multiply this by 104 to get the # cells/ml in the suspension.
If, however, the suspension was diluted prior to counting, then one would also multiply by the dilution factor to get the # cells/ml in the culture flask.
Avg. # cells counted x 104 = # cells/ml (x DF if any)

18. Counting Rule
Do not count cells touching
Bottom line
Right line

19. Cell counting Measurement Characteristics Methods Direct
Distinguish viable
and non-viable cell
- Simple, quick, cheap
- A small fraction of the
total cells from a cell
suspension
- Microscopic counting(Hemocytometer)
: Trypan blue, Crystal violet
- Electronic counters
: Coulter counter
Indirect
- Monolayer
- Immobilized in matrix
- Lactate dehydrogenase activity
- Functional assay
- DNA labeling assay

20. Example 1 ; Observation of cell death
A group of hepatoma cells exposed to a diffusing wave of digitonin. Intact cells (green) are damaged by digitonin, loose the green fluorescence and acquire red fluorescence of PI.

21. M e t h o d s
Clean hemocytometer & coverslip and wipe with 70% alcohol before use
Place coverslip on hemocytometer
Mix the cell suspension gently
Aliquot 0.1 ml cell suspensions
Add 0.1 ml (2-fold dilution), 0.3 ml (4-fold dilution) or 0.9 ml (10-fold dilution) trypan blue : appropriate range of cells to be counted
Draw a sample into a Pasteur pipette after mixing
Draw the cell suspension in to fill the chamber
Using a light microscope at low power, count the number of cells
Count the viable & non-viable cells in both halves of the chamber

22. Calculations
A = Vol. Of cell solution (ml)
B = Dilution factor in trypan blue
C = Mean number of unstained cells
D = Mean number of dead/stained cells
104 = Conversion of 0.1 mm3 to ml
(1) Total number of viable cells
A Ⅹ B Ⅹ C Ⅹ 104
(2) Total dead cell count
A Ⅹ B Ⅹ D Ⅹ 104
(3) To give a total cell count
Viable cell count + dead cell count
(4) % viability
(Viable cell count/Total cell count) Ⅹ 100

23. Example Dilution factor Vol. of CS Cell count Total viable cells
Vol. : Volume
CS : Cell Solution
TB : Trypan blue
Dilution factor
Vol. of CS
Cell count
Total viable cells
0.1 ml CS +
0.1 ml TB (2)
20 ml 23
20Ⅹ2Ⅹ23Ⅹ104 = 9.2 Ⅹ106 cells
0.3 ml TB (4)
15 ml //
15Ⅹ4Ⅹ23Ⅹ104 = 1.38 Ⅹ107 cells
0.9 ml TB (10)
10 ml
10Ⅹ10Ⅹ23Ⅹ104 = 2.3 Ⅹ107 cells

24. RBC Example… Aspirate blood to 0.5, then dilute to 101 DF = 200
Counted 500 erythrocytes in 5 small squares
RBC = rbc x 25 Sm Sq x DF (200)
5 Sm Sq mm3
= 5.00 x 106/mm3 (L)

25. Automated trypan blue method for optimal cell viability determination

26. Electric Cell Counting using a COULTER® COUNTER
Run results

27. Electric Cell Counting using a COULTER® COUNTER