342
Related Techniques
Preparation
of cell
suspension
Preparation
of the
!mocytometer
Cell loading
Procedure
1. Make a dissociated cell suspension from the culture to be examined
by repeated gentle pipetting.
2. Dilute the cell suspension with physiological solution or culture media so as to obtain a suitable cell density for counting. Record the dilution ratio (P).
1. Prepare the hemocytometer by wetting the edges very slightly, and
pressing the cover glass down over the grooves and counting area.
The appearance of interference patterns (Newton's ring: rainbow colors between the cover glass and slide, like those formed by oil on water) indicates that the cover glass is properly attached.
1. Add trypan blue solution to the diluted cell suspension in a volume
ratio of 1 : 1.
2. Draw up the cell suspension into a micropipette.
3. Discard five drops from the tip of the pipette.
4. Infuse approximately lO Jll cell suspension into the interstice between
the hemocytometer and cover glass. Avoid overfilling or underfilling
the chamber.
5. Wait until all the cells settle.
Counting 1. Count the unstained cells in a I-mm 2 area.
2. Repeat the procedure from step 2 of "Cell loading" to step 1 of"Counting" at least three times.
3. Calculate the average number of cells/mm2 (M).
Results
The cell density (number of cells/m!) is calculated by the formula
M x P x 20,000.
Subprotocol 2
Counting Cells Using a Grid Projected onto a Microscope Field
By installing an ocular grid micrometer into the eyepiece of an inverted
microscope, a square grid can be projected onto the field of a microscope. The real size corresponding to the projected square can be calibrated using an objective micrometer. Using this ocular grid, the number of cells in a culture vessel can be counted without disturbing the
culture.
Materials
Equipment - Inverted microscope
- Ocular micrometer (grid type)
Related Techniques
Preparation
of cell
suspension
Preparation
of the
!mocytometer
Cell loading
Procedure
1. Make a dissociated cell suspension from the culture to be examined
by repeated gentle pipetting.
2. Dilute the cell suspension with physiological solution or culture media so as to obtain a suitable cell density for counting. Record the dilution ratio (P).
1. Prepare the hemocytometer by wetting the edges very slightly, and
pressing the cover glass down over the grooves and counting area.
The appearance of interference patterns (Newton's ring: rainbow colors between the cover glass and slide, like those formed by oil on water) indicates that the cover glass is properly attached.
1. Add trypan blue solution to the diluted cell suspension in a volume
ratio of 1 : 1.
2. Draw up the cell suspension into a micropipette.
3. Discard five drops from the tip of the pipette.
4. Infuse approximately lO Jll cell suspension into the interstice between
the hemocytometer and cover glass. Avoid overfilling or underfilling
the chamber.
5. Wait until all the cells settle.
Counting 1. Count the unstained cells in a I-mm 2 area.
2. Repeat the procedure from step 2 of "Cell loading" to step 1 of"Counting" at least three times.
3. Calculate the average number of cells/mm2 (M).
Results
The cell density (number of cells/m!) is calculated by the formula
M x P x 20,000.
Subprotocol 2
Counting Cells Using a Grid Projected onto a Microscope Field
By installing an ocular grid micrometer into the eyepiece of an inverted
microscope, a square grid can be projected onto the field of a microscope. The real size corresponding to the projected square can be calibrated using an objective micrometer. Using this ocular grid, the number of cells in a culture vessel can be counted without disturbing the
culture.
Materials
Equipment - Inverted microscope
- Ocular micrometer (grid type)