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2 .Lab Exp - Hemocytometer

Hemocytometers are specialized slides used to count cells in suspension. They have a grid pattern that is visible under a microscope. To use one, a cell suspension is placed under a coverslip on the slide. Cells in squares of the grid are then counted to determine the concentration of cells per volume of suspension. Different types of cells or densities may require counting different areas of the grid. The cell concentration can be calculated based on the number counted, the volume of the grid area, and any dilution factors used. Hemocytometers are widely used for applications requiring quantification of cells, such as hematology, microbiology, and cell culture.

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Abhishek Kumar
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15 views

2 .Lab Exp - Hemocytometer

Hemocytometers are specialized slides used to count cells in suspension. They have a grid pattern that is visible under a microscope. To use one, a cell suspension is placed under a coverslip on the slide. Cells in squares of the grid are then counted to determine the concentration of cells per volume of suspension. Different types of cells or densities may require counting different areas of the grid. The cell concentration can be calculated based on the number counted, the volume of the grid area, and any dilution factors used. Hemocytometers are widely used for applications requiring quantification of cells, such as hematology, microbiology, and cell culture.

Uploaded by

Abhishek Kumar
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Dr.J.K.

Srivastava-AIB

Exp-1: Describe principle and application


Hemocytometer / Cell Counting Chamber/ Neubauer
chamber slide

For hematology, microbiology, cell culture, and many applications that require use of
suspensions of cells it is necessary to determine cell concentration. A device used for
determining the number of cells per unit volume of a suspension is called a counting
chamber. The most widely used type of chamber is called a hemocytometer, since it
was originally designed for performing blood cell counts. This is specially designed slide
having marking to form counting chambers , which can be viewed under microscope .

To prepare the counting chamber the mirror-like polished surface is carefully cleaned
with lens paper. The coverslip is also cleaned. Coverslips for counting chambers are
specially made and are thicker than those for conventional microscopy, since they must
be heavy enough to overcome the surface tension of a drop of liquid. The coverslip is
placed over the counting surface prior to putting on the cell suspension. The suspension
is introduced into one of the V-shaped wells with a pasteur or other type of pipet. The
area under the coverslip fills by capillary action. Enough liquid should be introduced so
that the mirrored surface is just covered. The charged counting chamber is then placed
on the microscope stage and the counting grid is brought into focus at low power.
Dr.J.K.Srivastava-AIB

Counting chamber

16 RBC Counting
Chambers

1 WBC Counting
Chamber

It is essential to be extremely careful with higher power objectives, since the counting
chamber is much thicker than a conventional slide. The chamber or an objective lens
may be damaged if the user is not not careful. One entire grid on standard
hemacytometers with Neubauer rulings can be seen at 40x (4x objective). The main
divisions separate the grid into 9 large squares (like a tic-tac-toe grid). Each square has
a surface area of one square mm, and the depth of the chamber is 0.1 mm. Thus the
entire counting grid lies under a volume of 0.9 mm-cubed.
Dr.J.K.Srivastava-AIB

Suspensions should be dilute enough so that the cells or other particles do not overlap
each other on the grid, and should be uniformly distributed. To perform the count,
determine the magnification needed to recognize the desired cell type. Now
systematically count the cells in selected squares so that the total count is 100 cells or
so (number of cells needed for a statistically significant count). For large cells this may
mean counting the four large corner squares and the middle one. For a dense
suspension of small cells you may wish to count the cells in the four 1/25 sq. mm
corners plus the middle square in the central square. Always decide on a specific
counting patter to avoid bias. For cells that overlap a ruling, count a cell as "in" if it
overlaps the top or right ruling, and "out" if it overlaps the bottom or left ruling.

Here is a way to determine a particle count using a Neubauer hemocytometer. Suppose


that you conduct a count as described above, and count 187 particles in the five small
squares described. Each square has an area of 1/25 mm-squared (that is, 0.04 mm-
squared) and depth of 0.1 mm. The total volume in each square is (0.04)x(0.1) = 0.004
mm-cubed. You have five squares with combined volume of 5x(0.004) = 0.02 mm-
cubed. Thus you counted 187 particles in a volume of 0.02 mm-cubed, giving you
187/(0.02) = 9350 particles per mm-cubed. There are 1000 cubic millimeters in one
cubic centimeter (same as a milliliter), so your particle count is 9,350,000 per ml.

Cells are often large enough to require counting over a larger surface area. For
example, you might count the total number of cells in the four large corner squares plus
the middle combined. Each square has surface area of 1 mm-squared and a depth of
0.1 mm, giving it a volume of 0.1 mm-cubed. Suppose that you counted 125 cells (total)
in the five squares. You then have 125 cells per 0.5 mm-cubed, which is 250 cells/mm-
cubed. Again, multiply by 1000 to determine cell count per ml (250,000).

Sometimes you will need to dilute a cell suspension to get the cell density low enough
for counting. In that case you will need to multiply your final count by the dilution factor.
For example, suppose that for counting you had to dilute a suspension of
Chlamydomonas 10 fold. Suppose you obtained a final count of 250,000 cells/ml as
Dr.J.K.Srivastava-AIB

described above. Then the count in the original (undiluted) suspension is 10 x 250,000
which is 2,500,000 cells/ml.

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