Procedure for Cell Counting

1.    Scope

·       This SOP explains counting of cells.

2.    Definition

• ml – milliliter
• GM –Growth Medium
• PBS – Phosphate Buffer Saline without Ca⁺⁺ and Mg⁺⁺.
• EDTA – Ethyl Di-amine Tetra acetate.

3.    Material

• Improved Neubauer Chamber
• Incomplete PBS (without Ca⁺⁺ and Mg⁺⁺)
• Trypsin EDTA
• 10% Growth Medium                                                     
• Trypan Blue
• Pipettes and tips
• Eppendorf tube

4.    Procedure

• Take 75/150cm² flask, which has a healthy monolayer.
• Take 0.3 ml trypan blue in an eppendorf tube.
• Wash the cells with 5ml/10ml PBS (without Ca⁺⁺ and Mg⁺⁺)
• Remove PBS from flask with pipette.
• Add 5ml/10ml trypsin –EDTA   into the flask in such a manner that it should cover the monolayer completely.
• Leave   it until streaks appear and then remove trypsin –EDTA using pipette.
• Add  5ml/10ml 10% GM and mix well with pipette to break clumps and take 0.1ml cell and add in to the trypan blue containing Eppendorf tube to make 1:4 dilution of cells.
• Mix well and charge Neubauer chamber on both sides using a pipette tip, avoid air bubble.

• Count unstained (viable) cells in each of the four corner squares bordered by triple lines, omitting cells lying on these lines. This is repeated for the second side of the chamber.
• Dead cells are stained blue by trypan blue, omit these cells in counting. If no of blue cells are more than unstained cells do not use that cell. Take another cell culture flask.
• Calculate the mean count of the total unstained (viable) cells per eight Squares.
• Calculate the unstained (viable) cell concentration per ml using the following
• Formula.

• C1                   =          t  × tb  ×1/4 ×10⁴
• t                       =          Total viable cell count of eight corner squares.
• 1/8                   =          for calculating mean cells per corner squares.
• tb                     =          dilution factor for trypan blue dilution.
• C₁                                 =          initial cell concentration per ml

Conversion Factor for Counting Chamber

• In Neubeaur chamber each large square has an area of 1 sq mm. when cover slip is placed  over the chamber is 0.1 mm, so volumes      =1 ×1 ×0.1

=0.1 cmm

If X cells in 0.1 cmm

So, X cells × 10 in 1 cmm

X cells × 10× 10³ IN 1 ml.

(1ml = X 10⁴/ml.)

1.    Safety conditions  

·       Handle Trypan Blue with gloved hands because it is carcinogenic.

·       Charge the Neubar chamber properly.

2.    Documentation 

·       Cell count register PR-36

3.    References used to draw SOP

• WHO Polio Laboratory Manual.