1. CELL COUNTING METHODS

2. CELL COUNTING HEMOCYTOMETER ELECTRONIC CELL COUNTER
AIM OF CELL COUNTING = to determine growth in cultures.
DIRECT METHODS
HEMOCYTOMETER
ELECTRONIC CELL COUNTER
INDIRECT METHODS
PROTEIN AND DNA ESTIMATION
GLUCOSE UPTAKE

3. HEMOCYTOMETER
Thick glass plate fits into stage of microscope . most common type is Neubauer cytometer.
grooved calibrated grid consist of 9 large squares with Imm side each. square further sub divided into 25 squares which is further subdivided into 16 squares of 0.05mm side.
cell suspension is put into the hemocytometer and viewed under microscope.
Each cell contain 0.1 µl of cell suspension.
Cell per ml = total count/ 5 x 10-4

4. Cell viability check by using TRYPAN BLUE DYE which permeates into non-viable cells.
mixture of citric acid and crystal violet causes cell to lysis and the released nuclei stains purple. used for viability measurement.
% of viability = no. of viable cells x 100
total cell count

6. ADVANTAGES
simple and effective.
DISADVANTAGES
Expected error is 10%.
Laborious method . If many samples being analysed.

7. ELECTRONIC CELL COUNTER
Electronic device which is suitable for cell counting rapidly.
0.5ml of cell suspension is diluted in PBS (phosphate buffer saline) and is pass through a small pore of 70µ in diameter.
Cell cause measureable change in electrical resistance as they passed between 2 electrodes. One inside and one outside the glass tube.
Pulses are recorded by oscilloscope. resistance produce is directly proportional to the volume of the cells.
The expected error is 5%.

9. ADVANTAGES
High speed for counting large no. of samples.
Expected error is less than 5%.
Particles smaller than cell can be eliminated from the count by setting lower threshold of detection.
DISADVANTAGES
Cell aggregates should not present in sample otherwise cell count will underestimated.
Cell suspension above (10)4 per ml should be diluted otherwise two or more cell passing through counting zone at the same time will causes error.

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