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Cell Counting Neubauer Chamber PDF
Cell Counting Neubauer Chamber PDF
Introduction Materials
Despite the fact of the recent technical The necessary elements to perform a cell count
development of scientific laboratories, the with Neubauer chamber are as follows:
Neubauer chamber remains the most common
method used for cell counting around the world. a) cellular dilution to measure
b) hemocytometer, or Neubabuer
This article has been written in order to help chamber
newbies and experimented researchers to c) optical microscope
perform a proper cell counting using a d) cover glass
Neubauer chamber or Hemocytometer. e) pippette / micropippete with
disposable tips.
The principles described in this article apply to f) Dilution buffer / PBS (if needed)
any cell counting chamber, although the
dimensions and volumes of each chamber may THE NEUBABUER CHAMBER, OR
differ. HEMOCYTOMETER
First, the parts and basic principle of the The Neubauer chamber is a thick crystal slide
Neubauer chamber are described with the size of a glass slide. (30 x 70 mm and
4 mm thickness)
Second, the article describes how to perform a
cell count step by step, in order to achieve In a simple counting chamber, the central area
reliable and reproducible results. The article is where cell counts are performed. The
describes best practices and recommendations chamber has three parts.
when performing a cell count.
c e f
a b
d
3
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Technical Note - Neubauer Chamber Cell Counting - Oscar Bastidas 2
The central part, where the counting grid has Neubauer chamber’s counting grid is 3 mm x 3
been set on the glass. mm in size. The grid has 9 square subdivisions
of width 1mm. (See Fig. 4-1)
Double chambers are most common than
simple chamber. In this case, the chamber has In case of blood cell counting, the squares
two counting areas than can be loaded placed at the corners are used for white cell
independently. counting. Since their concentration is lower than
red blood cells a larger area is required to
perform the cell count.
Upper chamber The central square is used for platelets and red
cells. This square is split in 25 squares of width
0,2 mm ( 200µm). See Fig. 4-2. Each one of the
25 central squares is subdivided in 16 small
squares. Fig 4-3.
GLASS COVER.
PIPETTE
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Technical Note - Neubauer Chamber Cell Counting - Oscar Bastidas 3
1 1
1 1
3
2
3 3
2 2
1 1
2
1
2 2
1 1 1
1 1
1
See:
http://www.celeromics.com/conteo-celular-con-
concentraciones-bajas.htm, for an statistical explanation
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Technical Note - Neubauer Chamber Cell Counting - Oscar Bastidas 4
STEP 2. Introducing the sample into the STEP 3. Microscope set up and focus.
Neubauer chamber
1. Place the Neubauer chamber on the
Take 10 µl of dilution prepare in STEP 1 with the microscope stage. If the microscope
micropipette. has a fixing clamp, fix the Neubauer
chamber.
1) Put the glass cover on the Neubauer 2. Turn on the microscope light.
chamber central area. Use a flat 3. Focus the microscope until you can
surface to place the chamber, like a see a sharp image of the cells looking
table or a workbench. through the eyepiece and adjusting
2) Put a disposable tip at the end of the the stage.
micropipette. 4. Look for the first counting grid square
3) Adjust the micropipette to suck 10 µl. where the cell count will start. In this
You can adjust it by turning the upper example, 5 big squares from a
plunger roulette to select the required Neubauer-Improved chamber will be
pipetting volume. counted. See Fig. 6
4) Introduce the micropipette tip on the
dilution previously prepared (STEP 1) See http://www.celeromics.com/easy-
5) Push the pipette plunger slowly until formula-for-manual-cell-counting.htm,
you feel it has arrived to the end of its for the formulas to be applied for the most
travel. common counting chamber (Thoma,
6) Remove the pipette tip from the Fuchs-Rosenthal, Nageotte, etc)
dilution, and bring it to the Neubauer
chamber. When the pipette is loaded, 5. Start counting the cells in the first
it must always be held in vertical square.
position.
7) Place pipette tip close to the glass Different laboratories have different
cover edge, right at the centre of the counting protocols, but there is a
Neubauer chamber. popular unwritten rule that states:
8) Release the plunger slowly watching
how the liquid enters the chamber “Cells touching the upper and left
uniformly, being absorbed by limits should be counted, unlike cells
capillarity. See Fig. 5 touching the lower and right limits
9) In case of the appearance of bubbles, which should not be taken into
or that the glass cover has moved, account”
repeat the operation.
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Technical Note - Neubauer Chamber Cell Counting - Oscar Bastidas 5
Number of cells
Concentration (cel / ml) = -------------------
Volume (in ml)
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Technical Note - Neubauer Chamber Cell Counting - Oscar Bastidas 6
Example:
For a 1 : 10 dilution. Dilution = 0,1
For a 1 : 100. Dilution = 0,01
Error
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error.htm
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