Download MEASUREMENT OF CELL COUNT AND VIABILITY

CELL COUNTING
AIM OF CELL COUNTING = to determine growth in cultures.
DIRECT METHODS
INDIRECT METHODS
HEMOCYTOMETER
PROTEIN AND DNA ESTIMATION
ELECTRONIC CELL COUNTER
GLUCOSE UPTAKE
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
 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
total cell count
x 100
 ADVANTAGES

simple and effective.
DISADVANTAGES

Expected error is 10%.

Laborious method . If many samples being analysed.
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%.
 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.