11

CELL COUNTING PROCEDURE

(1) Place the cover glass over the ruled area of a counting chamber. These have
special cover slips which allow correct depth of the chamber beneath. Do not
use ordinary cover slips for this purpose.

(2) With sterile technique and a sterile 1.0-ml pipet, remove 0.5 ml of well-
suspended cells from the graduate cylinder and place in a small test tube.

(3) With a fresh pipet, remove 1.0 ml of trypan blue stain (Appendix p. 102) from
its bottle, wipe the outside tip of the pipet with a Kimwipe tissue, and add the
stain to the cells in the tube.

(4) Mix the contents of the tube thoroughly by gently aspirating with a sterile pipet.
Remove a 0.5-ml sample.

(5) Quickly wipe the outside tip end of the pipet with a Kimwipe and place the tip
of the pipet to the edge of the cover slip on the counting chamber. Release the
pressure slightly on the mouth of the pipet, and allow the fluid to run into the
counting chamber. It may take a bit of practice to do this. You must allow the
fluid to fill only one side of the chamber. Do not add so much fluid that it flows
into the channels on each side of the counting area. It might be good practice to
try using colored water until you get the technique down to a point where you
feel comfortable doing it (Fig. 3-14).

(6) Allow the cells to settle for 2 min. Carefully lift the chamber and place it on the
microscope stage.

(7) With the low power objective in place, focus on the ruled area of the chamber.
The counting chamber actually has two ruled areas, one on each side of a
central trough (Fig. 3-14). One ruled area will be found to be sectioned like
this: (Figures 3-15)
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Fig. 3-14. Filling counting chamber.

Fig. 3-15. Counting chamber grid.

Focus your attention on the four corner blocks (marked 1, 2, 3, 4, above).

Each of these square millimeter areas is divided into 16 squares. With the
cover slip in place, the volume over one large square is 0.1 c. mm.

8) You will be concerned first with finding the average number of cells
per milliliter of your concentrate.
(a) Count the cells in several squares and obtain an average number
of cells per square. You may count only the four corner squares
on both ruled areas or, to increase your accuracy, count the
center squares also.
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EXAMPLE

Square No.

1 2 3 4 5 6 7 8 9 10
Count (No. of
cells per 36 40 38 41 * 39 43 41 34 *
square):
* Center square of each side not counted.

(b) Determine the average number of cells per square. From the example above
where eight squares were counted,

39 cells per square

8)312

(c) Adjust for the dilution. In preparing the materials for counting, you combined
0.5 ml of cell concentrate (one volume) with 1.0 ml (twice as much, or two
volumes) of stain diluent. You have, therefore, made a 1 + 2 (or I in 3, or 3X)
dilution of the concentrate. From step b above, you know you have 39 cells per
square of the stain-diluted concentrate. You must now compute how many
more cells there would have been had the material been counted undiluted. To
do this

Take the average number of cells per square: 39

Times the dilution of concentrate: 3
To get the total number of cells per 0.1 cubic ___
mm (millimeter) of concentrate 117

(d) Now, to correct this value to a count per milliliter of concentrate, you must
multiply by 10,000. The rationale is as follows:
1 ml = 1 cc (cubic centimeter).
1 cc is represented by a cube which is 1 cm (or 10 mm) on each edge.
1 cc has 10 x 10 x 10mm = 1000 cubic mm.
Our count of the average number of cells per square was based
on the volume of that square, i.e., 0.1 cubic mm.
To bring this to a 1-cm value, multiply by 10.
To bring the 1-cm value to I-mi value, multiply by 1000 or, combining the two,
multiply the average number of cells per square by 10,000. In brief, average
number of cells per square x dilution factor x 10,000 = number of cells per
milliliter of concentrate.
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(9) Enumerate all the cells with clear-cut nuclei and surrounding cytoplasm which appear in the
white cell (four corner squares) areas:
(a) Count single cells as one cell [Fig. 3-16(a)].
(b) Count clumps in which individual nuclei and cytoplasm are easily visible as clumps
of single cells, and count each cell [Fig. 3-16(b)].
(c) When individual cells are not easily discernible as such, clumps should be counted
as a single cell [Fig. 3-16(c)].

Fig. 3-16. Criteria for cell count.

Fig. 3-17. Path of cell counting.

(d) In counting the cells, develop the technique of counting from left to right on the
first row, right to left on the second row, left to right on the third row, right to
left on the fourth row (Fig. 3-17). Make it a matter of technique to include in
the count those cells which touch the inner line on the top and right-hand side
of the first and third rows and the top and left-hand side of the second and
fourth rows (Fig. 3-18).

Fig. 3-18. Cell Count.

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(10) Divide the total number of cells in all four corner squares by four to find the
average number of cells per square.
(11) The average number of cells per square times 10,000 (correction factor) times 3
(dilution factors) will give the cell count per milliliter, i.e., the number of cells in
each milliliter of fluid in the graduate cylinder.

J. DILUTION OF CELL SUSPENSION

Prepare several sets of tubes with cells varying in number from 150,000 per milliliter to
300,000 per milliliter. Individuals will vary in their counting technique, and that number of
cells which will produce a good cell sheet for one person will not be same for another. The type
of tissue will also determine the dilution. Cell concentration may vary between 2 and 8 x 105.
1. To determine the dilution factor (i.e., how much to dilute the concentrated cells in
order to obtain 150,000 or 300,000 cells per milliliter), divide the cell count per
milliliter determined above by the number of cells per milliliter desired.

EXAMPLE

Cells per milliliter of concentrate: 1,000,000

Cells per milliliter desired: 200,000
1,000,000 =5
200,000

This means that you must dilute the concentrated cells five times in order to

Fig. 3-19. Dilution of cell suspension.