Neubauer chamber or hemocytometer are more convenient for counting microbes. For microbiology, cell culture, and many applications that require use of suspensions of cells it is necessary to determine cell concentration. One can often determine cell density of a suspension spectrophotometrically, however that form of determination does not allow an assessment of cell viability, nor can one distinguish cell types.
The neubauer chamber is a heavy glass slide with two counting areas separated by a H-shaped through figure. 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.
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.
1 small box = 1mm / 4
= 0.25 mm
Average of cell numbers = 2 + 2 + 2 + 4 + 5 / 5
= 3
Volume of one small box = Area x Depth
= 0.25 mm x 0.25 mm x 0.1 mm
= 6.25 x 10 ⁻ᶟ mmᶟ
= 6.25 x 10 ⁻ᶟ x 10⁻ᶟ cmᶟ
= 6.25 x 10 ⁻⁶ cmᶟ
= 6.25 x 10 ⁻⁶ mL
Concentration of cells = average number of cells / volume
= 3 / 6.25 x 10⁻⁶ mL
= 480 000 cells/ mL
DISCUSSION
A device used for determining the number of cells per unit volume of a suspension is called a neubauer chamber. The most widely used type of chamber is called a hemocytometer, since it was originally designed for performing blood cell counts.
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.
Volume.(calculation)
square has surface area of 1 mm-squared and a depth of 0.1 mm
1.0mm ÷ 4 =0.25mm
Area of the square
Length × width = 0.25mm × 0.25mm
= 0.0625mm²
volume = area × depth
= 0.0625 mm² × 0.1 mm
= 6.25 × 10¯³ mm³
= 6.25 nm³
If measuring mitotic index (fancy name for % dividing cells), have a separate
clicker to count the number of cells dividing and just divide this by the total number of
algae counted (x100%).
replicate counts are depends on the amount of variability between counts and
the worse the technique, the more replicate chamber counts you will have to perform. It
also depends on how critical it is to get an accurate count.
REFERENCES ( for 1.1 and 1.2 )
academic.keystone.edu/JSkinner/Ocular%20Micrometer.doc
hi. this is the blog that have been created for IBG 102.
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