Cell counting

아래는 같은 실험실의 Joseph dela Cruz 가 작성한 레포트

 

 

Joseph dela Cruz

 

Lab Report #1

1.      Date:                   March 24, 2012

2.      Title:                   Cell Counting

3.      Purpose:             To quantify the concentration of cells in a suspension.

4.      Principle:           

The most common method for efficient and accurate counting of cells is with the use of haemocytometer.  The concentration of a cell suspension can be determined by allowing the cells to fill an optically flat chamber under a microscope. The cell number within the defined area of known depth is then counted and the cell concentration can now be derived from the count.

5.      Materials           

Sterile: 

Phosphate Buffer Solution A

Trypsin

DMEM growth media

Cell culture

                                    Nonsterile:

                           Tryphan blue stain dye

                           Pipettor

                           Hemocytometer

                           Tally counter

                           Microscope                                 

6.      Methods

a.      Trypsinize the monolayer, harvest cells, centrifuge and resuspend in DMEM medium.

b.      Clean the surface of the slide, taking care not to scratch the semi-silvered surface. Clean the cover slip and press it down, over the counting area of the slide.

c.       Mix the cell suspension thoroughly, pipetting vigorously and collect 10μL of cell suspension.

d.      Transfer the cell suspension immediately to the edge of the haemocytometer chamber and let the suspension fill the counting area of the slide. Do not overfill or underfill the chamber.

e.      Select the 10x objective of the microscope and focus on the grid lines in the chamber. Count the cells lying within the counting area (A,B,C,D)

f.        Count the number of cells in 4 corner squares and get the average. (Total cells counted/4). The depth of the chamber is 0.1 mm and assuming that only the central 1mm² is used, the volume is 0.1mm³, or 1 x 10⁴/ ml.  The concentration is then equal to average number of cells counted x 10⁴.

g.      Tryphan blue can also be used in the counting of cells to determine live from dead cells. For this method, collect 10μL of cell suspension and mix with 10μL of tryphan blue. Then aspirate 10μL and transfer immediately to the haemocytometer for counting. Count the number of cells in 4 corner squares and get the average. Divide the total cells counted by 4, then multiply by 2 to get the concentration (10⁴/ml)

7.     

Results

Method 1: Without dye

Cells counted: 21 cells

21/4= 5.25

Cell concentration= 5.25 x 10⁴/mL

 

Method 2: With Tryphan blue dye

Cells counted: 11 cells

(11/4) x 2 = 5.5

Cell concentration= 5.5 x 10⁴/ml

 

 

8.      Discussion

Haemocytometer counting is cheap and gives us the opportunity to see what we are counting. If the cells were mixed with a viability stain like Tryphan blue, a viability determination may be performed at the same time. However, this method is rather slow and prone to error.

Errors can happen during sampling, dilution, mixing and filling the chamber and by inaccurate counting. The following can cause inaccuracy of the haemocytometer method by affecting the volume of the chamber (Doyle and Griffiths, 1998):

a.                          Overflowing of the chamber

b.                          Incomplete filling of the chamber

c.                          Air bubbles and debris in the chamber

Also, make sure that the suspension is mixed well before taking a sample and do not allow the cells time to settle in the pipette tip before the transfer.

Other techniques and automatic methods of cell counting are also available.

 

9.      References

Culture of Animal Cells. A Manual of Basic Technique. 6^th edition.

R. Ian Freshney.

John Wiley and Sons, Inc., Publication, 2010. p335-338.

 

Cells and Tissue Culture: Laboratory Procedures in Biotechnology.

Edited by Alan Doyle and J. Bryan Griffiths

John Wiley and Sons, Inc., Publication, 1998. p57-61.