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Determination of Cell Count

Measuring the functionality of a medium, determining population doubling times, or tracking

the growth of cells in a culture, all require a means of quantifying cell population. The most
common method of determining cell number involves using a hemacytometer, an instrument
for estimating the number of cells in a measured volume under a microscope. Combined with
a vital stain, such as Trypan blue, the viability of the measured volume can also be
The improved Neubauer Hemacytometer
is a thick glass slide with two counting
chambers, each 0.1 mm deep. Each
chamber is divided into 9 large squares
delineated by triple white lines. The center
square is further divided into 25 squares.
These 25 squares are again subdivided
into 16 squares. The total surface area of
the hemacytometer is 9mm.
Hemacytometer cover slips must be used
to ensure even surfaces and coverage of
both counting chambers.

Shaded cells are counted as inside the

square counting area

Trypan blue is the most common stain used to distinguish viable cells from nonviable cells;
only non-viable cells absorb the dye and appear blue and may also appear asymmetrical.
Live, healthy cells appear round and refractive without absorbing the dye. The use of this
stain, however, is time sensitive. Viable cells absorb Trypan blue over time, so make dilutions
just prior to counting to avoid false data.

Determining cell density using a hemacytometer

The following procedure will be performed utilizing aseptic technique, as described in Aseptic
Techniques Used for Mammalian Cell Culture
1. For adherent cell lines, aspirate media and wash monolayer with PBS.
2. Aspirate PBS and trypsinize cell monolayer until all cells are detached.
3. For suspension cell lines begin at this step. Pipet all fluid and cell materials into a centrifuge
4. Weigh and balance tube within .5g and centrifuge at 1200rpm for seven minutes.
5. Aspirate the supernatant from the tube.
6. Prepare a cell suspension of the culture by adding media to the cell pellet and gently
o Use 10mL for T75, 5mL for T25, 2mL for 6-well, 1mL for 12-well and 24-well.
7. Using a microcentrifuge tube, make a 1:10 dilution of the cell suspension totaling 100L in
8. Wet the rails on the hemacytometer using a moist Kimwipe. Slide the cover glass on, ensuring
that the glass is firmly secured on the rails.
9. Vortex cell suspension and fill each chamber with the cell suspension using a pipette set to
10L. Dispense the solution smoothly in one motion, but not forcefully.
o If the solution spills into the grooves or there are bubbles covering the grid, clean the
hemacytometer with ethanol and water and repeat the application.
10. Place the hemacytometer on the stage of an inverted microscope and adjust the focus.
11. Use a hand-held counter to record cell counts in each of the four corners.
o Count cells touching the middle line of the triple line on the top and the left of the
squares. Do not count cells touching the middle line of the triple lines on the bottom or
right side of the square.
o If cells being counted are too small for the 10x view, then count cells in the central
square on a higher magnification.
12. Determine the number of cells per milliliter using the following calculation:

Determining cell viability using Trypan blue

The following procedure will be performed utilizing aseptic technique, as described in Aseptic
Techniques Used for Mammalian Cell Culture
1. Follow steps 1-7 in Determining cell density using a hemacytometer.
2. Create a 1:1 dilution of the cell suspension and 0.4% (w/v) Trypan blue by adding 100L
Trypan blue to the cell suspension.
3. Allow the Trypan blue to incubate for three minutes.
4. Follow steps 8-11 in Determining cell density using a hemacytometer.
a. All counting should be completed within 5 minutes of Trypan blue introduction to
minimize cell death and live cell absorption of dye.
b. When counting total both number of cells without dye and total number of cells.
5. Calculate the percentage of viable cells using the following formula: